Detection device for light transmitted from a sensed volume

ABSTRACT

A high-throughput light detection instrument and method are described. In some embodiments, switch mechanisms and optical relay structures permit different light sources and/or detectors to be selected for different applications. In other embodiments, switch mechanisms and optical paths permit top/bottom illumination and/or top/bottom detection, or combinations thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/160,533, filed Sep. 24, 1998, now U.S. Pat. No. 6,097,025, which isincorporated herein by reference.

This application is a continuation of the following patent applications,each of which is incorporated herein by reference: U.S. patentapplication Ser. No. 09/062,472, filed Apr. 17, 1998, now U.S. Pat. No.6,071,748; PCT Patent Application Serial No. PCT/US98/14575, filed Jul.15, 1998; U.S. patent application Ser. No. 09/118,141, filed Jul. 16,1998, now U.S. Pat. No. 6,313,960; U.S. patent application Ser. No.09/118,310, filed Jul. 16, 1998, now U.S. Pat. No. 6,033,100; U.S.patent application Ser. No. 09/118,341, filed Jul. 16, 1998, now U.S.Pat. No. 6,025,985; U.S. patent application Ser. No. 09/144,575, filedAug. 31, 1998, now U.S. Pat. No. 6,159,425; U.S. patent application Ser.No. 09/144,578, filed Aug. 31, 1998; U.S. patent application Ser. No.09/146,081, filed Sep. 2, 1998, now U.S. Pat. No. 6,187,267; U.S. patentapplication Ser. No. 09/156,318, filed Sep. 18, 1998, now U.S. Pat. No.6,258,326; and U.S. patent application Ser. No. 09/478,819, filed Jan.5, 2000.

This application is based upon and claims benefit under 35 U.S.C. § 119of the following U.S. Provisional Patent Applications, each of which isincorporated herein by reference: Ser. No. 60/052,876 filed Jul. 16,1997; Ser. No. 60/059,639 filed Sep. 20, 1997, Serial No. 60/063,811,filed Oct. 31, 1997; Serial No. 60/072,499, filed Jan. 26, 1998; SerialNo. 60/072,780, filed Jan. 27, 1998; Serial No. 60/075,414, filed Feb.20, 1998; Serial No. 60/075,806, filed Feb. 24, 1998; Serial No.60/082,253, filed Apr. 17, 1998; Serial No. 60/084,167, filed May 4,1998; Serial No. 60/085,335, filed May 13, 1998; Serial No. 60/085,500,filed May 14, 1998; Serial No. 60/089,848, filed Jun. 19, 1998; SerialNo. 60/094,275, filed Jul. 27, 1998; Serial No. 60/094,276, filed Jul.27, 1998; Serial No. 60/094,306, filed Jul. 27, 1998; Serial No.60/100,817, filed Sep. 18, 1998; and Serial No. 60/100,951, filed Sep.18, 1998.

FIELD OF THE INVENTION

The invention relates to instrumentation and methods for detectinglight. In particular, the invention relates to a versatile, sensitive,high-throughput screening apparatus that quantifies light transmittedfrom an assay site.

BACKGROUND OF THE INVENTION

High-throughput screening instruments are critical tools in thepharmaceutical research industry and in the process of discovering anddeveloping new drugs. The drug discovery process involves synthesis andtesting, or screening, of candidate drug compounds against a target. Acandidate drug compound is a molecule that might mediate a disease byits effect on a target. A target is a biological molecule, such as anenzyme, receptor, other protein, or nucleic acid, that is believed toplay a role in the onset or progression of a disease or a symptom of adisease. FIG. 1 shows stages of the drug discovery process, whichinclude target identification, compound synthesis, assay development,screening, secondary screening of hits, and lead compound screening, oroptimization, and finally clinical evaluation.

Targets are identified based on their anticipated role in theprogression or prevention of a disease. Until recently, scientists usingconventional methods had identified only a few hundred targets, many ofwhich have not been comprehensively screened. Recent developments inmolecular biology and genomics have led to a dramatic increase in thenumber of targets available for drug discovery research.

After a target is selected, a library of compounds is selected to screenagainst the target. Compounds historically have been obtained fromnatural sources or synthesized one at a time. Compound libraries werecompiled over decades by pharmaceutical companies using conventionalsynthesis techniques. Recent advances in combinatorial chemistry andother chemical synthesis techniques, as well as licensing arrangements,have enabled industrial and academic groups greatly to increase thesupply and diversity of compounds available for screening againsttargets. As a result, many researchers are gaining access to librariesof hundreds of thousands of compounds in months rather than years.

Following selection of a target and compound library, the compounds mustbe screened to determine their effect on the target, if any. A compoundthat has an effect on the target is defined as a hit. A greater numberof compounds screened against a given target results in a higherstatistical probability that a hit will be identified.

Prior to screening compounds against a target, a biological test orassay must be developed. An assay is a combination of reagents that isused to measure the effect of a compound on the activity of a target.Assay development involves selection and optimization of an assay thatwill measure performance of a compound against the selected target.Assays are broadly classified as either biochemical or cellular.Biochemical assays usually are performed with purified moleculartargets, which generally have certain advantages, such as speed,convenience, simplicity, and specificity. Cellular assays are performedwith living cells, which may sacrifice speed and simplicity, but whichmay provide more biologically relevant information. Researchers use bothbiochemical and cellular assays in drug discovery research.

Biochemical and cellular assays may use a variety of detectionmodalities, including photoluminescence, chemiluminescence, andabsorbance. Photoluminescence and chemiluminescence assays involvedetermining the amount of light that is emitted from excited electronicstates created by absorption of light and certain chemical reactions,respectively. Absorbance assays involve determining the amount of lightthat is transmitted through a composition relative to the amount oflight incident on the composition.

Each detection modality may use a variety of equipment. For example,photoluminescence assays typically employ at least a light source,detector, and filter; absorbance assays typically employ at least alight source and detector; and chemiluminescence assays typically employat least a detector. Moreover, the type of light source, detector,and/or filter employed typically varies even within a single detectionmodality. For example, among photoluminescence assays, photoluminescenceintensity and steady-state photoluminescence polarization assays may usea continuous light source, and time-resolved photoluminescencepolarization assays may use a time-varying light source.

Adding to this variability, the types of assays that are desired forhigh-throughput screening are evolving constantly. As new assays aredeveloped in research laboratories, tested, and published in literatureor presented at scientific conferences, new assays become popular andmany become available commercially. New analytical equipment may berequired to support the most popular commercially available assays.

After selection of a target, compound library, and assay, assays are runto identify promising compound candidates or hits. Once a compound isidentified as a hit, a number of secondary screens are performed toevaluate its potency and specificity for the intended target. This cycleof repeated screening continues until a small number of lead compoundsare selected. The lead compounds are optimized by further screening.Optimized lead compounds with the greatest therapeutic potential may beselected for clinical evaluation.

Due to the recent dramatic increase in the number of available compoundsand targets, a bottleneck has resulted at the screening stage of thedrug discovery process. Historically, screening has been a manual,time-consuming process. Recently, screening has become more automated,and standard high-density containers known as microplates have beendeveloped to facilitate automated screening. Microplates aresubstantially rectilinear containers that include a plurality of samplewells for containing a plurality of samples. Ninety-six-well microplateformats have been and still are commonly used throughout thehigh-throughput screening industry. However, some high-throughputscreening laboratories are using 384- and 768-well plates, and somelaboratories are experimenting with 1536-, 3456-, and 9600-wellmicroplates.

FIG. 2 shows a stack of overlapping microplates with various welldensities. Plate 30 has 96 wells. Plate 32 has 384 wells. Plate 34 has1536 wells. Plate 36 has 3456 wells. Plate 38 has 9600 wells. FIG. 2illustrates the substantial differences in well dimensions and densitiesthat may be used in high-throughput screening assays. Many analyzers arenot flexible enough to read microplates having different numbers ofwells, such that it currently may be necessary to provide differentanalyzers for different modes of analysis. Moreover, many analyzers arenot sensitive or accurate enough to read results from the smaller wellsassociated with the higher-density microplates. Inadequate sensitivitymay result in missed hits, limited research capabilities, increasedcosts of compounds, assays, and reagents, and lower throughput.

Screening an increasing number of compounds against an increasing numberof targets requires a system that can operate with a high degree ofautomation, analytical flexibility, and speed. In particular, becausehigh-throughput applications may involve repeating the same operationshundreds of thousands of times, even the smallest shortcomings aregreatly magnified. Current screening systems operate with variousdegrees of automation. Automation, from sample dispensing to datacollection, enables round-the-clock operation, thereby increasing thescreening rate. Automated high-throughput screening systems usuallyinclude combinations of assay analyzers, liquid handling systems,robotics, computers for data management, reagents and assay kits, andmicroplates.

Most analyzers in use today are not designed specifically forhigh-throughput screening purposes. They are difficult and expensive tointegrate into a high-throughput screening environment. Even after theanalyzer is integrated into the high-throughput screening environment,there often are many problems, including increased probability of systemfailures, loss of data, time delays, and loss of costly compounds andreagents.

In addition, most analyzers in use today offer only a single assaymodality, such as absorbance or chemiluminescence, or a limited set ofmodalities with non-optimum performance. To perform assays usingdifferent detection modes, researchers generally must switch single-modeanalyzers and reconfigure the high-throughput screening line.Alternatively, researchers may set up the high-throughput screening linewith multiple single-mode analyzers, which often results in criticalspace constraints.

Thus, prior detection devices generally have not recognized the need toprovide analytic flexibility and high performance for assay developmentas well as ease of use and smooth automation interface for thehigh-throughput screening laboratory. A real need exists for aversatile, sensitive, high-throughput screening apparatus that canhandle multiple detection modalities and wide ranges of sample volumesand variations in container material, geometry, size, and density formatwhile reliably maintaining a high level of sensitivity.

SUMMARY OF THE INVENTION

The present invention provides an apparatus and method for detectinglight transmitted from a composition. The apparatus and method mayemphasize plural light sources and/or detectors. The apparatus andmethod also may emphasize top/bottom illumination and/or detection.

In an embodiment emphasizing plural light sources, the apparatusincludes (1) a stage for supporting a composition at an examinationsite, (2) at least two different light sources and a first optical relaystructure that directs light from one of the light sources toward thecomposition, (3) a detector and a second optical relay structure thatdirects light from the composition toward the detector, and (4) a firstswitch mechanism that alters alignment of the first optical relaystructure from one of the light sources to another of the light sources,so that different light sources can be selected and directed toward theexamination site for different applications.

In an embodiment emphasizing plural detectors, the apparatus includes(1) a stage for supporting a composition at an examination site, (2) alight source and a first optical relay structure that directs light fromthe light source toward the composition, (3) at least two detectors anda second optical relay structure that directs light from the compositionto one of the detectors, and (4) a first switch mechanism that altersalignment of the second optical relay structure from one of thedetectors to another of the detectors, so that different detectors canbe selected for different applications.

In an embodiment emphasizing top/bottom illumination, the apparatusincludes (1) a stage for supporting a composition at an examinationsite, the examination site having a top side and a bottom side, (2) atleast one light source and a first optical relay structure defining afirst optical path directed toward the top side of the examination siteand a second optical path directed toward the bottom side of theexamination site, (3) at least one detector and a second optical relaystructure that directs light from the composition toward the detector,and (4) a first switch mechanism that alters alignment of the lightsource from one of the optical paths to the other optical path.

In an embodiment emphasizing top/bottom detection, the apparatusincludes (1) a stage for supporting a composition at an examination sitehaving a top side and a bottom side, (2) at least one light source and afirst optical relay structure that directs light from the light sourcetoward the composition, (3) at least one detector and a second opticalrelay structure defining a first optical path directed toward the topside of the examination site and a second optical path directed towardthe bottom side of the examination site, and (4) a first switchmechanism that alters alignment of the detector from one of the opticalpaths to the other optical path.

In yet other embodiments, light sources and detectors are replaced withadjacent compartments for light sources and detectors.

The apparatuses described above further may include (1) additional lightsources and detectors, (2) controllers preprogrammed to activate theswitch mechanisms for selecting light sources and detectors forparticular assays, (3) bar code readers for further automating thecontrollers, (4) filter alignment mechanisms for aligning filters withlight sources and detectors, (5) shuttles for aligning the optical relaystructures, and (6) automated registration devices for facilitatingsuccessive analysis of multiple compositions. The optical relaystructures further may include optical paths connecting light sourcesand detectors to top and bottom sides of the examination site to permit(1) top-illumination and top-detection, (2) top-illumination andbottom-detection, (3) bottom-illumination and top-detection, and (4)bottom-illumination and bottom-detection. Preferred light sourcesinclude high-intensity, high-color temperature arc lamps, and preferreddetectors include photomultiplier tubes.

The present invention also provides methods of detecting lighttransmitted from a composition.

In an embodiment emphasizing plural light sources, the method includes(1) providing a plurality of light sources, at least one detector, andan optical relay structure in a light detection instrument, wherein theoptical relay structure directs light from one of the light sourcestoward a composition at an examination site, (2) selecting one of thelight sources using a first switch mechanism that alters alignment ofthe optical relay structure from one of the light sources to another ofthe light sources, (3) relaying light from the selected light sourcethrough the optical relay structure to the composition, and (4)detecting light transmitted from the composition.

In an embodiment emphasizing plural detectors, the method includes (1)providing at least one light source, a plurality of detectors, and anoptical relay structure in a light detection instrument, wherein theoptical relay structure directs light from a composition at anexamination site toward one of the detectors, (2) selecting one of thedetectors using a first switch mechanism that alters alignment of thefirst optical relay structure from one of the detectors to another ofthe detectors, (3) illuminating the composition, and (4) relaying lightfrom the composition through the optical relay structure to the selecteddetector.

The methods described above further may involve selecting among bothlight sources and detectors, and/or among top/bottom illumination anddetection.

The nature of the invention will be understood more readily afterconsideration of the drawings and the detailed description of thepreferred embodiment that follow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow chart showing elements of the drug discovery process.

FIG. 2 is a top view of overlapping microplates showing variations inwell density.

FIG. 3 is a schematic view of analyzer components employed in anembodiment of the invention.

FIG. 4 is a schematic partial perspective view of analyzer componentsemployed in an embodiment of the invention.

FIG. 5 is a schematic view of optical components of a luminescenceoptical system employed in an embodiment of the invention.

FIG. 6 is a schematic view of optical components of a chemiluminescenceoptical system employed in an embodiment of the invention.

FIG. 7 is a cross-sectional perspective view of a top optics heademployed in an embodiment of the invention.

FIG. 8 is a cross-sectional perspective view of an alternative topoptics head employed in an embodiment of the invention.

FIG. 9 is a partially schematic cross-sectional view of achemiluminescence head employed in an embodiment of the invention.

FIG. 10 is a cross-sectional perspective view of a portion of thechemiluminescence head shown in FIG. 8.

FIG. 11 is a partial perspective view of top and bottom optics headsemployed in an embodiment of the invention.

FIG. 12 is a partially schematic side elevation view of the opticsassembly shown in FIG. 11, showing an offset between the top and bottomoptics head and side illumination.

FIGS. 13-16 are schematic views of sensed volumes in microplate wells.

FIG. 17 is a schematic top view of a microplate.

FIG. 18 is a graph showing the relationships between critical Z-heightand microplate well height.

FIG. 19 is a partial perspective, partial schematic view of a lightsource module employed in an embodiment of the invention.

FIG. 20 is a partial perspective view of an alternative light sourcemodule.

FIG. 21 is a partial perspective, partial schematic view of a detectormodule employed in an embodiment of the invention.

FIG. 22 is a partial perspective view of an alternative detector module.

FIG. 23 is a partial perspective view of a fiber optic shuttle assemblyemployed in an embodiment of the invention.

FIG. 24 is a perspective view of a floating head assembly employed inthe fiber optic shuttle assembly shown in FIG. 23.

FIG. 25 is a cross-sectional view of the floating head assembly, takengenerally along the line 25—25 in FIG. 24.

FIG. 26 is a perspective view of an alternative floating head assembly.

FIG. 27 is a cross-sectional view of the alternative floating headassembly, taken generally along the line 27—27 in FIG. 26.

FIG. 28 is a partially exploded perspective view of an optical filterwheel assembly employed in an embodiment of the invention.

FIG. 29 is a partially exploded perspective view of a portion of anoptical filter wheel assembly like that shown in FIG. 28, showing amechanism by which short filter cartridges may be removed.

FIG. 30 is a partially exploded perspective view of the portion of theoptical filter wheel assembly shown in FIG. 29, showing a mechanism bywhich tall filter cartridges may be removed.

FIG. 31 is a perspective view showing a mechanism by which opticalfilters may be placed in a tall filter cartridge.

FIG. 32 is a perspective view showing a mechanism by which a frictionmember may be pressed into place using a funnel and slug.

FIG. 33 is a top view of a short filter cartridge employed in anembodiment of the invention.

FIG. 34 is a cross-sectional view of the short filter cartridge, takengenerally along the line 34—34 in FIG. 33.

FIG. 35 is a top view of a tall filter cartridge employed in anembodiment of the invention.

FIG. 36 is a cross-sectional view of the tall filter cartridge, takengenerally along the line 36—36 in FIG. 35.

FIG. 37 is a top view of a funnel structure employed in conjunction withan embodiment of the invention.

FIG. 38 is a cross-sectional view of the funnel structure, takengenerally along the line 38—38 in FIG. 37.

FIG. 39 is a perspective view of a pivotable filter cartridge employedin an embodiment of the invention.

FIG. 40 is a perspective view of the top of a transporter assemblyemployed in an embodiment of the invention.

FIG. 41 is a perspective view of the bottom of the transporter assemblyshown in FIG. 40.

FIG. 42 is a partial cross-sectional view of the transporter assemblyshown in FIGS. 40 and 41, taken generally along the line 42—42 in FIG.41.

FIG. 43 is a perspective view of a base platform and associated drivemechanisms for moving a transporter along X and Y axes relative to thebase platform.

FIG. 44 is a partially exploded perspective view of a housing for ananalyzer constructed in accordance with the invention.

FIG. 45 is a front view of the control unit shown in FIG. 44.

FIG. 46 is a top view of one of the control interface docking locationsshown in FIG. 44.

FIG. 47 is a front view of the input/output panel shown in FIG. 44.

FIG. 48 is a perspective view of a sample feeder constructed inaccordance with the invention, with bins removed so that internalmechanisms of the sample feeder can be viewed.

FIGS. 49A and 49B are cross-sectional views through a first (input)station of the sample feeder shown in FIG. 48, taken generally along theline 49AB—49AB in FIG. 48 and showing latch and lifter cooperation toremove a microplate from the bottom of a stack.

FIGS. 50A and 50B are cross-sectional views through a third (output)station of the sample feeder shown in FIG. 48, taken generally along theline 50AB—50AB in FIG. 48 and showing latch and lifter cooperation toadd a microplate to the bottom of a stack.

FIG. 51 is a side elevation view of a lifter from the sample feedershown in FIG. 48.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an analyzer capable of supporting a wide range ofassay formats that can be carefully selected and fine-tuned forscreening desired targets with flexibility, durability, and convenience.Flexibility means that the analyzer can be used with a variety ofsamples and sample assays. Durability means that the analyzer can beused repeatedly, at high throughput, in laboratory and industrialsettings. Convenience means that the analyzer can be used with onlyminimal user intervention, while also allowing assays to be run insmaller containers with reduced volumes.

The analyzer achieves these and other objectives, in part, by employingan optical system that minimizes sample interfacial boundaryinterference, thereby permitting reduction in assay volume in existingformats such as 96 or 384 well plates, and utilization of denser formatssuch as 768, 1536, 3456, or 9600 well plates. The analyzer also achievesthese objective, in part, by providing the ability automatically toswitch between different modes, including absorbance, photoluminescence,photoluminescence polarization, time-resolved photoluminescence,photoluminescence lifetime, and chemiluminescence modalities, amongothers.

The apparatus of the present invention generally includes a stage forsupporting a composition in an examination site, an automatedregistration device for bringing successive compositions and theexamination site into register for analysis of the compositions, a lightsource for delivering light into the compositions, a detector forreceiving light transmitted from the compositions, and an optical relaystructure for transmitting light substantially exclusively from a sensedvolume that may comprise only a portion of the composition.

Description of the Optical System

FIGS. 3-6 show a preferred embodiment of the optical system of ananalyzer 50 constructed in accordance with the present invention. Theoptical system generally includes at least one light source fordelivering light to a composition, at least one detector for receivinglight transmitted from the composition, and an optical relay structurefor relaying light between the light source, composition, and detector.The optical system may limit detection to a sensed volume that maycomprise only a portion of the composition.

Components of the optical system are chosen to optimize sensitivity anddynamic range for each assay mode supported by the analyzer. Toward thisend, optical components with low intrinsic luminescence are chosen. Inaddition, some components are shared by different modes, whereas othercomponents are unique to a particular mode. For example,photoluminescence intensity and steady-state photoluminescencepolarization modes share a light source; time-resolved luminescencemodes use their own light source; and chemiluminescence modes do not usea light source. Similarly, photoluminescence and chemiluminescence modesuse different detectors.

These assay modes all involve detection of luminescence, which is theemission of light from excited electronic states of atoms or molecules.Luminescence generally refers to all kinds of light emission, exceptincandescence, and may include photoluminescence, chemiluminescence, andelectrochemiluminescence, among others. In photoluminescence, includingfluorescence and phosphorescence, the excited electronic state iscreated by the absorption of electromagnetic radiation. Inchemiluminescence, which includes bioluminescence, the excitedelectronic state is created by a transfer of chemical energy. Inelectrochemiluminescence, the excited electronic state is created by anelectrochemical process.

Separate descriptions of the photoluminescence and chemiluminescenceoptical systems are presented below. Selected components of both systemsare described in greater detail in subsequent sections. The opticalsystem presented here is a preferred embodiment. The present inventionalso includes other arrangements and components capable of detectinglight from a sensed volume in high-throughput applications.

Photoluminescence Optical System

FIGS. 3-5 show the photoluminesnce optical system of analyzer 50.Because photoluminescence follows the absorption of light, thephotoluminescence optical system must include one or more light sources.In analyzer 50, there are two light sources. A continuous source 100provides light for photoluminescence intensity and steady-statephotoluminescence polarization assays. A preferred continuous source isa high-intensity, high-color temperature xenon arc lamp. The preferredsource provides more light per unit time than flash sources, increasingsensitivity and reducing read times. A time-modulated source 102provides light for time-resolved photoluminescence assays, such asphotoluminescence lifetime and time-resolved photoluminescencepolarization assays. A preferred time-modulated source is a xenon flashlamp. The preferred source produces a “flash” of light for a briefinterval before signal detection and is especially well suited fortime-domain measurements. Other time-modulated sources include pulsedlasers, as well as continuous lamps whose intensity can be modulatedextrinsically using a Pockels cell, Kerr cell, or other mechanism. Thelatter sources are especially well suited for frequency-domainmeasurements. Analyzer 50 includes light source slots 103 a-d for fourlight sources, although other numbers of light source slots and lightsources also could be provided. The direction of light transmissionthrough the photoluminescence optical system is indicated by arrows.

More generally, light sources include any sources of electromagneticradiation of any wavelength capable of inducing photoluminescence orabsorption in a composition. For example, light includes but is notlimited to ultraviolet, visible, and infrared radiation. Suitable lightsources include lamps, electroluminescence devices, lasers,light-emitting diodes (LEDs), and particle accelerators. Depending onthe source and assay mode, light produced by such light sources maybe 1) mono- or multichromatic, 2) polarized or unpolarized, 3) coherentor incoherent, and/or 4) continuous or time-modulated.

In analyzer 50, continuous source 100 and time-modulated source 102produce multichromatic, unpolarized, and incoherent light. Continuoussource 100 produces substantially continuous illumination, whereastime-modulated source 102 produces time-modulated illumination. Lightfrom these light sources may be delivered to the sample withoutmodification, or it may be filtered to alter its intensity, spectrum,polarization, or other properties.

Light produced by the light sources follows an excitation optical pathto an examination site. Such light may pass through one or more“spectral filters,” which generally comprise any mechanism for alteringthe spectrum of light that is delivered to the sample. Spectrum refersto the wavelength composition of light. A spectral filter may be used toconvert white or multichromatic light, which includes light of manycolors, into red, blue, green, or other substantially monochromaticlight, which includes light of one or only a few colors. In analyzer 50,spectrum is altered by an excitation interference filter 104, whichselectively transmits light of preselected wavelengths and selectivelyabsorbs light of other wavelengths. For convenience, excitationinterference filters 104 may be housed in an excitation filter wheel106, which allows the spectrum of excitation light to be changed byrotating a preselected filter into the optical path. Spectral filtersalso may separate light spatially by wavelength. Examples includegratings, monochromators, and prisms.

Spectral filters are not required for monochromatic (“single color”)light sources, such as certain lasers, which output light of only asingle wavelength. Therefore, excitation filter wheel 106 may be mountedin the optical path of some light source slots 1031 a,b, but not otherlight source slots 103 c,d.

Light next passes through an excitation optical shuttle (or switch) 108,which positions an excitation fiber optic cable 110 a,b in front of theappropriate light source to deliver light to top or bottom optics heads112 a,b, respectively. The optics heads include various optics fordelivering light into the sensed volume and for receiving lighttransmitted from the sensed volume. Light is transmitted through a fiberoptic cable much like water is transmitted through a garden hose. Fiberoptic cables can be used easily to turn light around corners and toroute light around opaque components of the analyzer. Moreover, fiberoptic cables give the light a more uniform intensity profile. Apreferred fiber optic cable is a fused silicon bundle, which has lowautofluorescence. Despite these advantages, light also can be deliveredto the optics heads using other mechanisms, such as mirrors.

Light arriving at the optics head may pass through one or moreexcitation “polarization filters,” which generally comprise anymechanism for altering the polarization of light. Polarization refers tothe direction of the electric field associated with light. Excitationpolarization filters may be included with the top and/or bottom opticshead. In analyzer 50, polarization is altered by excitation polarizers114, which are included only with top optics head 112 a. Excitationpolarization filters 114 may include an s-polarizer S that passes onlys-polarized light, a p-polarizer P that passes only p-polarized light,and a blank O that passes substantially all light. Excitation polarizers114 also may include a standard or ferro-electric liquid crystal display(LCD) polarization switching system. Such a system is faster and moreeconomical than a mechanical switcher. Excitation polarizers 114 alsomay include a continuous mode LCD polarization rotator with synchronousdetection to increase the signal-to-noise ratio in polarization assays.

Light at one or both optics heads also may pass through an excitation“confocal optics element,” which generally comprises any mechanism forfocusing light into a “sensed volume.” In analyzer 50, the confocaloptics element includes a set of lenses 117 a-c and an excitationaperture 116 placed in an image plane conjugate to the sensed volume, asshown in FIG. 5. Lenses 117 a,b project an image of this aperture ontothe sample, so that only a preselected or sensed volume of the sample isilluminated.

Light traveling through the optics heads is reflected and transmittedthrough a beamsplitter 118, which delivers reflected light to acomposition 120 and transmitted light to a light monitor 122. Reflectedand transmitted light both pass through lens 117 b, which is operativelypositioned between beamsplitter 118 and composition 120. Thebeamsplitter is changeable, so that it may be optimized for differentassay modes or compositions. The light monitor is used to correct forfluctuations in the intensity of light provided by the light sources;such corrections are performed by reporting detected intensities as aratio over corresponding times of the luminescence intensity measured bythe detector to the excitation light intensity measured by the lightmonitor. The light monitor also can be programmed to alert the user ifthe light source fails. A preferred light monitor is a siliconphotodiode with a quartz window for low autofluorescence.

The composition (or sample) is held in a sample container supported by astage 123. The composition can include compounds, mixtures, surfaces,solutions, emulsions, suspensions, cell cultures, fermentation cultures,cells, tissues, secretions, and/or derivatives and/or extracts thereof.Analysis of the compositions may involve measuring the presence,concentration, or physical properties of a photoluminescent analyte insuch a composition. The sample container can include microplates, genechips, or any array of samples in a known format. In analyzer 50, thepreferred sample container is a microplate 124, which includes aplurality of microplate wells 126 for holding compositions. Compositionmay refer to the contents of a single microplate well, or severalmicroplate wells, depending on the assay.

The position of the sensed volume within the composition created by theconfocal optics element can be moved precisely to optimize thesignal-to-noise and signal-to-background ratios. In analyzer 50,position in the X,Y-plane perpendicular to the optical path iscontrolled by moving the stage supporting the composition, whereasposition along the Z-axis parallel to the optical path is controlled bymoving the optics heads using a Z-axis adjustment mechanism 130, asshown in FIGS. 3 and 4. However, any mechanism for bringing the sensedvolume into register or alignment with the appropriate portion of thecomposition also may be employed.

The combination of top and bottom optics permits assays to combine: (1)top illumination and top detection, or (2) top illumination and bottomdetection, or (3) bottom illumination and top detection, or (4) bottomillumination and bottom detection. Same-side illumination and detection(1) and (4) is referred to as “epi” and is preferred forphotoluminescence assays. Opposite-side illumination and detection (2)and (3) is referred to as “trans” and is preferred for absorbanceassays. In analyzer 50, epi modes are supported, so the excitation andemission light travel the same path in the optics head. However, transmodes also could be supported and would be essential for absorbanceassays. Generally, top optics can be used with any sample containerhaving an open top, whereas bottom optics can be used only with samplecontainers having optically transparent bottoms, such as glass or thinplastic bottoms.

Light is transmitted by the composition in multiple directions. Aportion of the transmitted light will follow an emission pathway to adetector. Transmitted light passes through lens 117 c and may passthrough an emission aperture 131 and/or an emission polarizer 132. Inanalyzer 50, the emission aperture is placed in an image plane conjugateto the sensed volume and transmits light substantially exclusively fromthis sensed volume. In analyzer 50, the emission apertures in the topand bottom optical systems are the same size as the associatedexcitation apertures, although other sizes also may be used. Theemission polarizers are included only with top optics head 112 a. Theemission aperture and emission polarizer are substantially similar totheir excitation counterparts.

Excitation polarizers 114 and emission polarizers 132 may be usedtogether in nonpolarization assays to reject certain background signals.Luminescence from the sample container and from luminescent moleculesadhered to the sample container is expected to be polarized, because therotational mobility of these molecules should be hindered. Suchpolarized background signals can be eliminated by “crossing” theexcitation and emission polarizers, that is, setting the angle betweentheir transmission axes at 90°. To increase signal level, beamsplitter118 should be optimized for reflection of one polarization andtransmission of the other polarization. This method will work best wherethe luminescent molecules of interest emit relatively unpolarized light,as will be true for small luminescent molecules in solution.

Transmitted light next passes through an emission fiber optic cable 134a,b to an emission optical shuttle (or switch) 136. This shuttlepositions the appropriate emission fiber optic cable in front of theappropriate detector. In analyzer 50, these components are substantiallysimilar to their excitation counterparts, although other mechanisms alsocould be employed.

Light exiting the fiber optic cable next may pass through one or moreemission “intensity filters,” which generally comprise any mechanism forreducing the intensity of light. Intensity refers to the amount of lightper unit area per unit time. In analyzer 50, intensity is altered byemission neutral density filters 138, which absorb light substantiallyindependent of its wavelength, dissipating the absorbed energy as heat.Emission neutral density filters 138 may include a high-density filter Hthat absorbs most incident light, a medium-density filter M that absorbssomewhat less incident light, and a blank O that absorbs substantiallyno incident light. These filters are changed by hand, although othermethods also could be employed, such as a filter wheel. Intensityfilters also may divert a portion of the light away from the samplewithout absorption. Examples include beam splitters, which transmit somelight along one path and reflect other light along another path, andPockels cells, which deflect light along different paths throughdiffraction.

Light next may pass through an emission interference filter 140 whichmay be housed in an emission filter wheel 142. In analyzer 50, thesecomponents are substantially similar to their excitation counterparts,although other mechanisms also could be employed. Emission interferencefilters block stray excitation light, which may enter the emission paththrough various mechanisms, including reflection and scattering. Ifunblocked, such stray excitation light could be detected andmisidentified as photoluminescence, decreasing the signal-to-backgroundratio. Emission interference filters can separate photoluminescence fromexcitation light because photoluminescence has longer wavelengths thanthe associated excitation light.

The relative positions of the spectral, intensity, polarization, andother filters presented in this description may be varied withoutdeparting from the spirit of the invention. For example, filters usedhere in only one optical path, such as intensity filters, also may beused in other optical paths. In addition, filters used here in only topor bottom optics, such as polarization filters, may also be used in theother of top or bottom optics or in both top and bottom optics. Theoptimal positions and combinations of filters for a particularexperiment will depend on the assay mode and the composition, amongother factors.

Light last passes to a detector, which is used in absorbance andphotoluminescence assays. In analyzer 50, there is one photoluminescencedetector 144, which detects light from all photoluminescence modes. Apreferred detector is a photomultiplier tube (PMT). Analyzer 50 includesdetector slots 145 a-d for four detectors, although other numbers ofdetector slots and detectors also could be provided.

More generally, detectors comprise any mechanism capable of convertingenergy from detected light into signals that may be processed by theanalyzer. Suitable detectors include photomultiplier tubes, photodiodes,avalanche photodiodes, charge-coupled devices (CCDs), and intensifiedCCDs, among others. Depending on the detector and assay mode, suchdetectors may be used in (1) photon-counting or continuous modes, and(2) imaging or integrating modes.

Chemiluminescence Optical System

FIGS. 3, 4, and 6 show the chemiluminescence optical system of analyzer50. Because chemiluminescence follows a chemical event rather than theabsorption of light, the chemiluminescence optical system does notrequire a light source or other excitation optical components. Instead,the chemiluminescence optical system requires only selected emissionoptical components. In analyzer 50, a separate lenslesschemiluminescence optical system is employed, which is optimized formaximum sensitivity in the detection of chemiluminescence.

Generally, components of the chemiluminescence optical system performthe same functions and are subject to the same caveats and alternativesas their counterparts in the photoluminescence optical system. Thechemiluminescence optical system also can be used for other assay modesthat do not require illumination, such as electrochemiluminescence.

The chemiluminescence optical path begins with a chemiluminescentcomposition 120 held in a sample container 126. The composition andsample container are analogous to those used in photoluminescenceassays; however, analysis of the composition involves measuring theintensity of light generated by a chemiluminescence reaction within thecomposition rather than by light-induced photoluminescence. A familiarexample of chemiluminescence is the glow of the firefly.

Chemiluminescence light typically is transmitted from the composition inall directions, although most will be absorbed or reflected by the wallsof the sample container. A portion of the light transmitted through thetop of the well is collected using a chemiluminescence head 150, asshown in FIG. 3, and will follow a chemiluminescence optical pathway toa detector. The direction of light transmission through thechemiluminescence optical system is indicated by arrows.

The chemiluminescence head includes a nonconfocal mechanism fortransmitting light from a sensed volume within the composition.Detecting from a sensed volume reduces contributions to thechemiluminescence signal resulting from “cross talk,” which is pickupfrom neighboring wells. The nonconfocal mechanism includes achemiluminescence baffle 152, which includes rugosities 153 that absorbor reflect light from other wells. The nonconfocal mechanism alsoincludes a chemiluminescence aperture 154 that further confinesdetection to a sensed volume.

Light next passes through a chemiluminescence fiber optic cable 156.This fiber optic cable is analogous to excitation and emission fiberoptic cables 110 a,b and 134 a,b in the photoluminescence opticalsystem. Fiber optic cable 156 may include a transparent, open-endedlumen that may be filled with fluid. This lumen would allow the fiberoptic to be used both to transmit luminescence from a microplate welland to dispense fluids into the microplate well. The effect of such alumen on the optical properties of the fiber optic could be minimized byemploying transparent fluids having optical indices matched to theoptical index of the fiber optic.

Light next passes through one or more chemiluminescence intensityfilters, which generally comprise any mechanism for reducing theintensity of light. In analyzer 50, intensity is altered bychemiluminescence neutral density filters 158. Light also may passthrough other filters, if desired.

Light last passes to a detector, which converts light into signals thatmay be processed by the analyzer. In analyzer 50, there is onechemiluminescence detector 160. This detector may be selected tooptimize detection of blue/green light, which is the type most oftenproduced in chemiluminescence. A preferred detector is a photomultipliertube, selected for high quantum efficiency and low dark count atchemiluminescence wavelengths (400-500 nanometers).

Optics Heads and the Generation of Sensed Volumes

FIG. 7 shows a cross-sectional view of top optics head 112 a, which isused together with fiber optic cables 110 a, 134 a and apertures 116,131, as shown in FIG. 5, to create the sensed volume. Top optics head112 a is substantially similar to bottom optics head 112 b, as shown inFIGS. 11 and 12, except that top optics head 112 a includeschemiluminescence head 150 and excitation and emission polarizers 114,132 (not shown), and that bottom optics head 112 b includes a window anddrip lip (described below).

Excitation light arrives at top optics head 112 a through excitationfiber optic cable 110 a. Fiber optic cables are cylindrical waveguidesthat transmit light through a process known as total internalreflection. Fiber optic cables are characterized by a numericalaperture, which describes the maximum angle through which the fiberoptic cable can collect light for total internal reflection. The higherthe numerical aperture, the greater the angle over which the fiber opticcable can collect and transmit light. The numerical aperture is definedas NA=n sin θ, where NA is the numerical aperture, n is the index ofrefraction of the medium adjacent the fiber optic cable, and θ is thehalf angle of the cone of transmitted or incident light. In top opticshead 112 a, the medium adjacent the fiber optic cable is air, so n≡1.

Excitation light exits fiber optic cable 110 a through excitationaperture 116 at a cone angle θ₁ determined in part by the numericalaperture of the fiber optic cable. In top optics head 112 a, exitingexcitation light forms a first cone 170 of excitation light, with itsapex positioned just inside the tip 172 of fiber optic cable 110 a.First cone 170 of excitation light passes through an excitationpolarizer 114 (not shown), and then through a first plano-convexconverging lens 174, whose plan side 176 is oriented toward fiber opticcable 110 a. First lens 174 is positioned so that it substantiallyconverts first cone 170 of excitation light into a first cylinder 178 ofexcitation light. This conversion is accomplished by positioning tip 172substantially at the focal point of first lens 174.

First cylinder 178 of excitation light impinges on beamsplitter 118 a.Beamsplitter 118 a reflects a reflected cylinder portion 180 ofexcitation light toward composition 120 in sample well 126. Reflectedcylinder portion 180 passes through a second plano-convex converginglens 182, whose plan side 184 is oriented away from beamsplitter 118 a.Second lens 182 converts reflected cylinder portion 180 of excitationlight into a second cone 186 of excitation light, which is focused ontoand thus delivered to composition 120 in sample well 126. The cone angleθ₂ of second cone 186 is determined in part by the numerical aperture ofsecond lens 182, and may be different from the cone angle θ₁ describingexcitation light exiting fiber optic cable 110 a.

Beamsplitter 118 a also transmits a transmitted cylinder portion 188 ofthe excitation light to light monitor 122, which functions as describedabove. The optics used to focus the transmitted light into the lightmonitor may be substantially similar to the optics used to focus thereflected light into the sample well. Alternatively, the optics mayinclude a lensless system, such as a black tapered cone to direct light.

The excitation light may induce photoluminescence within thecomposition. Photoluminescence (or emission) light has longerwavelengths than the associated excitation light. This is due toconservation of energy; in photoluminescence, the emission light haslower energy (and so longer wavelength) than the excitation light,because some of the energy of the excitation light is lostnonradiatively.

A conical portion of the emission light substantially coextensive withsecond cone 186 of excitation light passes back through second lens 182,which converts the conical portion into a cylindrical portion ofemission light substantially coextensive with reflected cylinder 180 ofexcitation light.

Emission light next impinges on beamsplitter 118 a, which transmits acylinder portion 190 of emission light toward photoluminescence detector144. Beamsplitter 118 a typically is chosen to accommodate one of twodifferent scenarios. If a large number or variety of luminescentmolecules are to be studied, the beamsplitter must be able toaccommodate light of many wavelengths; in this case, a “50:50”beamsplitter that reflects half and transmits half of the incident lightindependent of wavelength is optimal. Such a beamsplitter can be usedwith many types of molecules, while still delivering considerableexcitation light onto the composition, and while still transmittingconsiderable emission light to the detector. If one or a few relatedluminescent molecules are to be studied, the beamsplitter needs only tobe able to accommodate light at a limited number of wavelengths; in thiscase, a “dichroic” or “multichroic” beamsplitter is optimal. Such abeamsplitter can be designed for the appropriate set of molecules andwill reflect most or substantially all of the excitation light, whiletransmitting most or substantially all of the emission light. This ispossible because the reflectivity and transmissivity of the beamsplittercan be varied with wavelength.

Cylinder portion 190 of emission light transmitted through beamsplitter118 a passes through a third plano-convex converging lens 192, whoseplan side 194 is oriented away from the beamsplitter. In first opticshead 112 a, emission light first may pass through an emission polarizer132, as shown in FIG. 5. Third lens 192 focuses the cylindrical portion190 of emission light into a third cone of light 196 that impinges onemission fiber optic cable 134 a for transmission to photoluminescencedetector 144. To be transmitted by the fiber, the light should befocused onto emission aperture 131 at the tip 198 of the fiber as a spotcomparable in size to the diameter of the fiber optic cable. Moreover,the incident cone angle θ₃ should not exceed the inverse sine of thenumerical aperture of the fiber.

A property of the optical arrangement in top optics head 112 a is thatthe tips 172, 198 of fiber optic cables 110 a, 134 a and the sensedvolume of the composition are “confocal.” Confocal means that all threeobjects are in conjugate focal planes, so that whenever one is in focus,all are in focus. The sensed volume of the composition lies in a focalor sample plane FP of the system, and the tips of the fiber optic cableslie in image planes IP of the system. The detector also may be placed inan image plane, so that it detects the composition in focus. The tips ofthe fiber optic cables may be said to lie in intermediate image planes,because light passes through these planes, and the detector may be saidto lie in a terminal image plane, because light terminates on thedetector.

The sensed volume is created by placing confocal optics elements in ornear one or more intermediate image planes. A preferred confocal opticselement is an aperture. If such an aperture is placed in the excitationoptical path, an image of the aperture will be focused onto thecomposition. As a result, only a portion of the composition within thefocal plane corresponding to the shape and proportional to the size ofthe aperture will be illuminated, and only luminescent molecules in ornear that portion of the focal plane will be induced to emitphotoluminescence. If such an aperture is placed in the emission opticalpath, an image of the aperture will be focused onto the detector.Luminescence that ordinarily would focus onto a part of the detectoroutside the image of the aperture will be blocked or masked fromreaching the detector.

The “shape” (or intensity profile) of the sensed volume depends on theconfocal optics elements, such as excitation and emission apertures 116,131, the light source, and the numerical apertures of the lenses andfiber optic cables. Generally, the intensity of the light incident on(or emitted from) the sensed volume will be greatest at the center ofthe sensed volume, and will decay monotonically in all directions awayfrom the center. Most of the intensity will lie within a distance equalto about one aperture diameter from the center of the sensed volume inthe Z direction, and within about one-half an aperture diameter from thecenter of the sensed volume in the X and Y directions.

FIG. 7 also shows a sample container sensor switch 230, which is used toprevent damage to optics head 112 a by preventing the optics head fromphysically contacting a sample container. Sample container sensor switch230 is mounted about a pivot axis P adjacent chemiluminescence head 150.Sample container sensor switch 230 includes a sensor surface 232positioned so that a sample container must contact the sensor surfacebefore contacting any component of top optics head 112 a. Contactbetween a sample container and sensor surface 232 causes samplecontainer sensor switch 230 to pivot about pivot axis P, activating anelectrical circuit that turns off power to the mechanism(s) used to movethe sample container.

A sample container sensor switch is especially important in an analyzerdesigned for use with a variety of sample containers, because it reducesthe likelihood of damage both from exotic sample holders with unusualdimensions and from standard sample holders with aberrant ormisidentified dimensions. The sample container sensor switch may detectimpending contact between the sample container and optics head (1)mechanically, as in the preferred embodiment, (2) optically, as with anelectric eye, (3) acoustically, as with an ultrasonic detector, or (4)by other mechanisms. For example, the sample container sensor switch mayinclude a linear voltage displacement transducer (LVDT), which measuresdisplacement by creating a voltage proportional to the displacement.

FIG. 7 also shows a chemiluminescence head 150, which includes achemiluminescence baffle 152 and a chemiluminescence fiber optic cable156. Chemiluminescence head 150 is mounted on top optics head 112 a, butalso could be mounted on bottom optics head 112 b or on both top andbottom optics heads 112 a,b.

FIG. 8 shows an alternative embodiment of top optics head 112 a, whichincludes an alternative embodiment of chemiluminescence head 150.

FIG. 9 shows an alternative view of chemiluminescence head 150. Inchemiluminescence, emission light sensitivity is maximized by detectingas much emission light as possible from the top of the sample container.In analyzer 50, this is accomplished by placing fiber optic cable 156directly above and aligned with the center of the microplate well orother sample container. A high numerical aperture fiber optic cable maybe used to collect most or substantially all of the light emitted fromthe composition. A preferred fiber optic cable has a numerical apertureof 0.22 and is formed of silica for low autoluminescence.

Detection of chemiluminescence light further is enhanced by positioningfiber optic cable 156 so that the gap G or flying height between thefiber optic cable and the top of the sample container is as small aspossible. Generally, if the gap between the top of the microplate andthe fiber optic cable is small compared to the diameter of the fiberoptic cable, most of the emission light will be collected. In analyzer50, preferred values of G lie in the range 0.25-1.5 mm, depending on thetype of microplate. The preferred values allow for normal variations inmicroplate thickness and minimize the possibility of contacting liquidthat may be on the surface of the microplate. This is accomplished byaccurate calibration of the travel of the optical head along the Z-axisrelative to a reference point on the Z-axis. The height of variousmicroplates can be stored in software so that G can be set by theinstrument to a preselected value.

Gap G also can be determined empirically using a precision top-of-platesensor, which is mounted on the bottom of the upper optics head. Theheight of the plate is measured by slowly moving the optics head towardthe plate until the top-of-plate sensor indicates that a known flyingheight has been achieved. With this approach, the height of the plateneed not be known in advance. Moreover, if a microplate mistakenly isinserted into the machine with a greater than expected height, thetop-of-plate sensor can be used to prevent the optics head fromcolliding with the microplate.

Chemiluminescence head 150 also includes a chemiluminescence baffle 152,which supports fiber optic cable 156 and an aperture support slide 250and which also minimizes detection of ambient light andchemiluminescence from neighboring wells. Detection from neighboringwells may be referred to as “cross talk.” In analyzer 50,chemiluminescence baffle 152 is generally circular and includes a blacksurface 252 with rugosities 153 designed to absorb light.Chemiluminescence baffle 152 may have a diameter at least about twicethe diameter of the fiber optic cable, and may be configured to allowlow cross talk to be achieved at comfortable flying heights.

FIG. 10 shows a partially cross-sectional perspective view ofchemiluminescence head 150. Chemiluminescence head 150 includes a fiberoptic cable 156 and an aperture support plate 250 containing apertures254 a,b that determine an “effective” entrance diameter for the fiberoptic cable. In turn, the effective entrance diameter for the fiberoptic cable determines the size of the sensed volume within the sample.To maximize signal, apertures 254 a,b generally are chosen substantiallyto equal the diameter of the microplate well. Large apertures 254 ahaving diameters larger than fiber optic cable 156, and small apertures254 b having diameters smaller than fiber optic cable 156 may be placedin front of the fiber optic cable. A moveable aperture support slide 250may include separate apertures for 96, 384, 768, 1536, 3456, and 9600well plates, among others, where each aperture is optimized for the wellsize associated with a particular microplate. Alternatively, a fixedaperture support slide 250 may include a continuous iris diaphragmaperture, where the size of the continuous diaphragm may be optimizedfor a range of well sizes.

Alternative embodiments of the chemiluminescence optical system couldinclude a plurality of chemiluminescence heads optically connected to aplurality of chemiluminescence detectors. The chemiluminescence headscould be mounted as a linear array or as a matrix. For example, a lineararray of 8 or 12 chemiluminescence heads optically connected to 8 or 12detectors could be used to detect simultaneously from entire rows orcolumns of a 96-well microplate. Moreover, the same arrays also could beused with the appropriate apertures to detect from higher-density platesin which the well-to-well spacing is evenly divisible into thewell-to-well spacing in the 96-well plate, as for 384 and 1536-wellplates. The chemiluminescence heads also could be mounted as a matrixthat could detect from one or more plate formats.

Other alternative embodiments of the chemiluminescence optical systemcould include a plurality of fiber optic cables connected as a bundle toa CCD detector or to a PMT array. The fiber optic bundle could beconstructed of discrete fibers or of many small fibers fused together toform a solid bundle. Such solid bundles are commercially available andeasily interfaced to CCD detectors.

These alternative embodiments may be used with alternative embodimentsof chemiluminescence baffle 152. For example, with a fiber optic bundle,cross-talk between wells within the matrix can be minimized by keeping Gas small as possible and/or by applying an anti-reflective coating tothe face of the fiber bundle. An anti-reflective coating can reducereflected light from about 4% to less than 1%. In addition, a bafflehaving a rough black surface as described above could be placed aroundthe outside of the fiber bundle, like a collar, to minimize pick-up fromareas of the plate that are not under the bundle.

FIG. 11 shows the relationship between top and bottom optics heads 112a,b and chemiluminescence head 150. Top and bottom optics heads 112 a,bare coupled to an optics head support structure 260, which includes agap 262 through which a stage and sample container can pass. Optics headsupport structure 260 is configured so that the relative positions oftop and bottom optics heads 112 a,b are fixed.

FIG. 11 also shows a Z-axis adjustment mechanism 130, which is used toadjust the position of a sensed volume within a composition. Z-axisadjustment mechanism 130 includes a support track 264 that issubstantially parallel to a Z-axis on which optics head supportstructure 260 is mounted. Z-axis adjustment mechanism 130 also includesa motor 266 for moving optics head support structure 260 along supporttrack 264. The position of a sensed volume within a compositionpositioned in gap 262 is adjusted by moving top and bottom optics heads112 a,b relative to the composition. Movement relative to thecomposition may be effected by moving the optics heads while keeping thecomposition stationary, as here, or by moving the composition whilekeeping the optics heads stationary, among other mechanisms.

FIG. 11 also shows aspects of bottom optics head 112 b. Generally,bottom optics head 112 b resembles top optics head 112 a. However,bottom optics head 112 b includes a window 267 and an elevated drip lip268 that are not included on top optics head 112 a. Window 267 and driplip 268 prevent fluid dripped from a microplate from entering bottomoptics head 112 b. Fluid dripped from a microplate is a concern withbottom optics head 112 b because the bottom optics head is positionedbelow the microplate during analysis.

FIGS. 11 and 12 show further aspects of bottom optics head 112 b.Generally, light is directed through bottom optics head 112 b much likelight is directed through top optics head 112 a. However, light also maybe directed by an alternative optical relay structure 269 to the bottom(or top) optics head. Alternative optical relay structure 269 mayinclude a fiber optic cable 270 and focusing lens structure 271.Off-axis illumination eliminates loss of light due to absorption andreflection from the beam splitter and substantially eliminatesreflection of incident light into the detection optics, reducingbackground. Off-axis illumination also may be used for total internalreflection illumination.

FIGS. 11 and 12 also show the relative positions of top and bottomoptics heads 112 a,b. Top and bottom optics heads 112 a,b may bealigned, so that excitation light transmitted by one optics head can bedetected by the other optics head, facilitating absorbance assays. Ashutter may be positioned between the two optics heads to prevent lightfrom one optics head from entering and exciting fluorescence from theother optics head during luminescence assays. Alternatively, top andbottom optics head 112 a,b may be offset, so that light from one opticshead cannot enter the other optics head. A small optical relaystructure, such as a fiber optic cable, may be positioned adjacent or aspart of bottom optics head 112 b to detect light in a top illuminationand bottom detection mode.

Application of Sensed Volumes

The optical system described above, and the confocal optics elements inparticular, allow detection of luminescence substantially exclusivelyfrom a sensed volume of a composition.

FIG. 13 shows a standard microplate well 126 and an excitation lightbeam 186 as it illuminates the well. The standard well is cylindricaland may be characterized by a diameter D_(W) and a height H_(W). Otherwells may have other geometries and be characterized by otherquantities; for example, a well could be square and characterized by awidth and a height, or a well could be conical and characterized by acone angle and a height. The interface between composition 120 and theair 272 is termed the meniscus 274 and may be convex, plan, or concave.

Excitation light beam 186 is focused by the optical system so that it isshaped much like an hourglass along the optical (Z) axis. This hourglassshape arises as the cone of excitation light formed by the optics passesthrough focus. The diameter D_(B) of the beam is smallest at the beam'swaist 276, which corresponds to the focal plane, above and below whichthe beam diverges monotonically, making an angle θ_(B) with respect tothe vertical or Z-axis. Values of D_(B) and θ_(B) depend on opticalcomponents of the analyzer and may be varied by changing thesecomponents. Generally, D_(B) and θ_(B) are inversely related. Thedistance between the bottom of the well and the beam waist is termed thefocal (Z) height, H_(Z).

The shape of the sensed volume, indicated by stippling, may differ indirections parallel and perpendicular to the optical or Z-axis. Parallelto the Z-axis, the shape may be Lorentzian, among others. Perpendicularto the Z-axis, the shape may be Gaussian, or it may be a rounded pulsefunction, among others. A laser beam might give rise to a Gaussian,whereas a fiber optic bundle might give rise to a rounded pulsefunction. Generally, lower numerical apertures will create sensedvolumes shaped more like cylinders, whereas higher numerical apertureswill create sensed volumes shaped more like hourglasses.

The shape and volume of the sensed volume may be adapted like a probe tomatch the shape and volume of the sample container. Thus, the sensedvolume may be expanded for maximum signal in a large sample container,and contracted to avoid nearby walls in a small sample container. Theshape and volume of the sample container also may be chosen or designedto conform to the shape and volume of the sensed volume.

Alternatively, the sensed volume may be held constant. In this way, thesensed volume will report on equal volumes of each composition analyzed,so that the analyzer effectively reports “intensive” quantities.Intensive quantities do not depend on the amount of composition in asample container; in contrast, extensive quantities do depend on theamount of composition in the sample container. This approach can be usedto facilitate comparison of results obtained from different-sized samplewells, such as in 96 and 384 well microplates. Alternatively, thisapproach can be used to facilitate comparison of results obtained fromlike-sized sample wells containing different volumes of solution, as bydesign or by error.

FIG. 14 shows how the signal-to-noise and signal-to-background ratiosare affected by focal height for two assay modes. In homogeneous assays(Panel B), photoluminescent molecules are distributed uniformlythroughout the composition, and the optimum signal-to-noise andsignal-to-background ratios are obtained regardless of well geometrywhen the sensed volume is positioned in the middle of the composition(Panel A), so that the sensed volume does not overlap with the meniscusor the bottom or sides of the well. If the meniscus is in the sensedvolume, light reflected from the meniscus will be detected. This willdecrease sensitivity by increasing background and decreasing signal. Ifthe bottom of the well is in the sensed volume, light reflected from thewell bottom will be detected. Moreover, noncomposition photoluminescencearising from fluorescent and other photoluminescent materials that arecommonly included in the microplate or adsorbed to the walls of themicroplate also will be detected. These two effects will decreasesensitivity by increasing background and decreasing signal. Luminescencemeasured from the microplate walls will lead to spuriously highluminescence intensities and luminescence polarizations.

In cell-based assays (Panels C and D), photoluminescent molecules areconcentrated in or near cells growing at the bottom of the well, and theoptimum signal-to-noise and signal-to-background ratios are obtainedwhen the sensed-volume is centered about the bottom of the well (PanelA). Such centering may be accomplished either using top optics (Panel C)or bottom optics (Panel D).

The shape and position of the sensed volume within the well are affectedby (1) the meniscus, (2) the geometry of the microplate well, and (3)the geometry of the whole microplate.

FIG. 15 shows how the meniscus affects the shape and position of thesensed volume. When there is no fluid and hence no meniscus, the beamhas a nominal undistorted shape; see Panel A. The meniscus affects thesensed volume because light is refracted as it crosses the meniscusboundary between the air and the composition. Specifically, lightpassing from air (with its lower index of refraction) to the composition(with its higher index of refraction) bends toward the normal, asdescribed by Snell's law. Here, the normal is the directionperpendicular to the surface of the meniscus at a given point. If themeniscus is everywhere perpendicular to the light beam, then lightpassing through the meniscus will not bend, and the beam will retain itsnominal undistorted shape. For a converging beam, this will occur whenthe meniscus is appropriately convex; see Panel B. If the meniscus ismore than appropriately convex, light will bend toward the middle of thewell as it passes through the meniscus, and the sensed volume will becompressed and raised; see Panel C. If the meniscus is less thanappropriately convex, flat, or concave, light will bend away from themiddle of the well as it passes through the meniscus, and the sensedvolume will be stretched and lowered; see Panel D. Meniscus effectscould be minimized by appropriately configuring microplate wells.

FIGS. 16 and 17 show how the geometry of the microplate well affects theposition of the sensed volume. In particular, if the well issufficiently narrow relative to the diameter of the beam or if the wellis sufficiently deep relative to the angle made by the beam, then thelight beam may impinge upon the top walls of the well. In these cases,setting the Z-height too low can reduce sensitivity (1) by decreasingthe desired signal because less light enters the well, and (2) byincreasing the background because the light beam illuminates the tops ofwells. Many microplates are made from materials that are fluorescent orotherwise photoluminescent, and the instrument will detect thisphotoluminescence from materials at the tops of wells.

FIG. 17 shows how the geometry of the microplate affects the position ofthe sensed volume. The analyzer is configured automatically to find thelocation of each well in a given microplate, beginning with well A1. Theanalyzer does this using stored parameters describing the dimensions(plate heights, interwell distances, etc.) of the particular microplatestyle. However, these microplate parameters are nominal values and donot account for unit-to-unit or lot-to-lot variations in microplategeometry. If there is a slight variation in interwell distance, thelight beam can be off-center on some wells even though it is perfectlycentered on well A1. This effect is termed cross-plate drift.

Cross-plate drift of fluorescence readings may increase as theinstrument scans across the microplate as variations are compounded.Typically, drift will be worst at well H12, which is farthest from wellA1. Such drift can be reduced by making the stage more accurate, bymaking the sample containers of a more consistent size, or by increasingH_(Z), which will reduce the diameter of the beam and put it back intothe well. The lattermost approach is shown for well G11.

Because beam position is a critical determinant of signal to noise, Zheight must be appropriately maintained; Z height should be kept above acritical focal height, H_(Z,Crit). The height at which the beam firstimpinges on the walls of the well is the critical focal height,H_(Z,Crit). FIG. 18 shows how H_(Z,Crit) depends on the well heightH_(W) and well diameter D_(W), for a beam of diameter 1.5 millimeters(mm) and a beam angle θ_(B) of 12.7 degrees. Similarly, Table 1 showshow H_(Z,Crit) depends on well height and well diameter for fourcommercially available microplates.

Well Well Height Diameter H_(Z,Crit) Plate Type (mm) (mm) (mm) CostarBlack Flat Bottom 96-Well 3915 10.71 6.71 −0.85 Dynatech MicroFluorRound Bottom 9.99 6.78 −1.72 Costar Black 384-Well 3710 11.55 3.66 6.76Packard White 384-Well #6005214 11.57 3.71 6.67

Z-height can be optimized for a particular microplate and chemistry by(1) preparing a test microplate with representative chemistry (e.g.,blanks, positive and negative controls, dilution series), (2) andreading the microplate multiple times at different Z-heights todetermine the Z-height that gives the best signal-to-background data.Some combinations of chemistry and microplate are relatively insensitiveto Z-height, while others demonstrate a distinct optimum.

As described above, a sample container sensor switch is mounted on thetop optics head to prevent the plate from contacting the optics head incase the plate is misaligned, not properly specified, or the Z-height isset incorrectly. If this sensor detects a fault, the sample containerwill be ejected prior to reading.

Although this discussion was presented for microplates, the sameprinciples apply with other sample containers.

Light Source and Detector Modules

FIG. 19 is a perspective view of a light source module 400 employed inan embodiment of the invention. Portions of the module case have beenremoved to reveal internal componentry. Light source module 400 includesat least two light sources. A flashlamp 402 transmits light along afirst light path 404. A second light source, namely, a continuous arclamp (not shown) housed in compartment 406, transmits light along asecond light path 408. A filter wheel assembly 410 is positionedadjacent the light sources. Filter wheel assembly 410 includes a filterwheel 412, which holds a plurality of filters 414. Filter wheel 412 isrotatable around an axis 416, so that a given filter can be positionedinterchangeably along light path 404, or along light path 408, byrotating filter wheel 412. A fiber optic shuttle assembly 418 is mountednext to filter wheel assembly 410. Moveable shuttle 420 translates alongsupport tracks 422 a and 422 b, so that moveable shuttle 420 can bepositioned in front of a selected light source for a selected assayapplication. Two fiber optic ports 424 are provided on an external faceof shuttle 420. Fiber optic ports 424 direct light, via fiber opticcables, from a selected source either to a top optics head or to abottom optics head, above and below a stage holding a sample,respectively.

FIG. 20 is a perspective view of an alternative light source module 426.In this embodiment, filter wheel assembly 410 of light source module 400has been replaced by an alternative filter wheel assembly 427. Amoveable shuttle 428 is shown in an alternative position relative tomoveable shuttle 420 in light source module 400.

FIG. 21 is a perspective view of a detector module 440 employed in anembodiment of the invention. Portions of the module case have beenremoved to reveal internal componentry. Detector module 440 is similarto light source module 400. A detector 442 receives light directed alonga light path 444, originating from a sample. A filter wheel assembly 446is positioned in front of detector 442. Filter wheel assembly 446includes a plurality of filters 450 and is rotatable around an axis 451by a stepper, DC servo, or other motor. The filter wheel can be rotatedat a preselected angular speed to allow synchronization with a flashlamp light source and a detector. A port 452 for a second detector isprovided in filter wheel assembly 446, so that a second detector can bemounted in detector module 440. A given filter in filter wheel 448 canbe positioned along a first light path 444 leading to detector 442, oralternatively can be positioned along a second light path leading to asecond detector (not shown). An attenuator mechanism 454 is mountedadjacent filter wheel assembly 446. A fiber optic shuttle assembly 456is mounted in front of attenuator mechanism 454. Shuttle assembly 456includes a moveable shuttle 458, which is moveable along upper and lowersupport tracks 460 a and 460 b, respectively. An exterior face ofshuttle 458 has two fiber optic ports 462, one of which is connected,via a fiber optic cable, to a top optics head above the examinationsite, the other of which is connected, via a fiber optic cable, to abottom optics head below the examination site. In operation, moveableshuttle 458 can be moved along support tracks 460 a and 460 b to connectoptically either one of the optics heads to any one of the detectors (ifmore than one is included in the module), and through any one of filters450 in filter wheel 448.

FIG. 22 is a perspective view of an alternative detector module 466. Inthis embodiment, filter wheel assembly 446 of detector module 440 hasbeen replaced by an alternative filter wheel assembly 467. A moveableshuttle 468 is shown in an alternative position relative to moveableshuttle 458 in detector module 440.

Light source and detector modules are designed for flexibility.Additional ports for fiber optics or other optical relay structures maybe provided, if desired. The number and configuration of such otherports may be tied to the number and configuration of light-transmissionroutes through the filter wheel. Optical components also may beconnected directly to the moveable shuttle. Such a connection would beespecially useful for small, dedicated components, such as abeamsplitter and photodiode-type detector that could sample a portion ofthe light transmitted through the port to correct for outputfluctuations from a light source.

A comparison of FIGS. 19 and 21, and FIGS. 20 and 22, shows that manyaspects of light source modules 400 and 426 and detector modules 440 and466 are the same, particularly the mechanics of filter wheel assemblies410 and 446, filter wheel assemblies 427 and 467, and fiber opticshuttle assemblies 418 and 456. The light source and detector modulesboth function as registration mechanisms that align the end of anoptical relay structure with an aperture in a surface. This surface mayenclose a light source, detector, or other optical component. The lightsource and detector modules both permit alignment with two suchapertures, and with portions of a surface not including an aperture toprevent the optical relay structure from transmitting light. Lightsource and detector modules also may be configured to transmit lightdirectly from module to module, using air, a tube, or other mechanism totransmit light. If used together in a light detection device, the lightsource and detector modules provide a great deal of analyticalflexibility to select different combinations of light sources,detectors, and filters for different applications, while also being ableto select different combinations of top versus bottom illumination anddetection orientations.

FIG. 23 is a partial perspective view of a fiber optic shuttle assembly480 like those used in light source module 400 and detector module 440.Fiber optic shuttle Among other applications, each floating headassembly 482 may be used to create and maintain a light-tight connectionbetween selected light sources or detectors and fiber optic cables, suchas those that lead to an examination site, or to a top optics head or abottom optics head, above and below a stage, respectively.

FIG. 24 shows a perspective view of a floating head assembly 483employed in an embodiment of the invention. Generally, floating headassembly 483 includes a fiber optic ferule 484 having an end 485configured to transmit light, and an opaque collar 486 positioned aroundthe end. Fiber optic ferule 484 is used to transmit light. Fiber opticferule 484 may be replaced by a portion of a light source, detector, orother optical component. Opaque collar 486 is used to block light andpreferably comprises a hard plastic material. Opaque collar 486encompasses and extends beyond end 485. An opaque base structure 487contains additional elements. Together, opaque collar 486 and basestructure 487 form a pair of concentric, partially overlapping wallspositioned around fiber optic ferule 484.

FIG. 25 is a cross-sectional view of floating head assembly 483. Aspring 488 is positioned between portions of opaque collar 486 and basestructure 487. Spring 488 generally comprises any elastic body or otherdevice that recovers its original shape when released after beingdistorted. Spring 488 is configured to spring-bias opaque collar 486relative to end 485 when spring 488 is compressed between opaque collar486 and base structure 487. Spring 488 bias pushes opaque collar 486 andbase structure 487 in opposite directions parallel to a central axis 489running through fiber optic ferule 484. A flange 490 on opaque collar486 contacts a retaining ring 491 on base structure 487 when opaquecollar 486 is maximally extended, limiting relative movement of opaquecollar 486 and base structure 487. Additional or alternative stopmechanisms also may be employed, such as a set screw.

In use, floating head assembly 483 is positioned such that fiber opticferule 484 is aligned with an aperture 492 in a surface 493, so thatlight may be transmitted between fiber optic ferule 484 and aperture492. When end 485 and aperture 492 are aligned, a leading rim edge 494of opaque collar 486 is spring-biased or forced against surface 493 bycompression of spring 488. Leading rim edge 494 defines an end planethat is moveable relative to central axis 489. Opaque collar 486 andthus leading rim edge 494 automatically float or reorient relative tosurface 493, forming a substantially light-tight junction by changingangle relative to central axis 489. This substantially light-tightjunction substantially prevents stray light from entering the system,and it substantially prevents signal light from exiting the system.Spring 488 is relatively more compressed where surface 493 is closer tofloating head assembly 483 and relatively less compressed where surface493 is farther from floating head assembly 483, so that contact betweenopaque collar 486 and surface 493 is maintained for different positionsand/or orientations of surface 493. Portions of opaque collar 486 may beformed of a material that deforms under pressure from spring 488 toconform substantially to asperities or other irregularities in surface493.

FIG. 26 shows a perspective view of an alternative floating headassembly 495. Generally, alternative floating head assembly 495 includesa fiber optic cable 496 having an end 497 configured to transmit light,and an opaque collar 498 positioned around the end.

FIG. 27 shows a cross-sectional view of alternative floating headassembly 495. Fiber optic ferule 496 and opaque collar 498 are supportedby a base structure 499 that includes a spherical bearing 500 having aninner race 501 and an outer race 502. Inner race 501 is slidinglyconnected to a sleeve portion 503 of opaque collar 498 that extendsalong fiber optic ferule 496. Outer race 502 is connected to a platformstructure 504 used for mounting alternative floating head assembly 495.A spring 505 is positioned between portions of opaque collar 498 andouter race 502. Spring 505 bias pushes opaque collar 498 and basestructure 499 in opposite directions parallel to a central axis 490running through fiber optic ferule 496. A retaining ring 507 preventsover-extension of opaque collar 498.

In use, alternative floating head assembly 495 is positioned, likefloating head assembly 483, such that fiber optic ferule 496 is alignedwith an aperture 508 in a surface 509, so that light may be transmittedbetween fiber optic ferule 496 and aperture 508. When so aligned, opaquecollar 498 and fiber optic ferule 496 are free to compress and extenddue to the action of spring 505, and to swivel and reorient due to theaction of spherical bearing 500, relative to surface 509. The combinedactions of spring 505 and spherical bearing 500 ensure that central axis506 of fiber optic ferule 496 always is substantially parallel to anaperture axis 510 running through aperture 508, unlike with floatinghead assembly 483.

Filter Wheel Assemblies

FIG. 28 shows a partially exploded perspective view of an optical filterwheel assembly 520 employed in an embodiment of the invention. Opticalfilter wheel assembly 520 includes a filter wheel 521 that is rotatableabout a hub structure 522, and a wheel case having a static base portion523 and a removable lid portion 524. Hub structure 522 is built intoremovable lid portion 524.

Filter wheel 521 holds filter cartridges 525. Filter wheel 521 issubstantially circular and includes a plurality of apertures 526disposed symmetrically about its outer perimeter 527. Apertures 526 areused for mounting filter cartridges 525 and may hold the filtercartridges via friction, threads, or other means. Filter wheel 521 mayhave a variety of shapes, and apertures 526 may be disposed in a varietyof configurations, although a symmetric embodiment is preferred forbalance and ease of rotation about hub structure 522.

Removable lid portion 524 holds filter wheel 521. Removable lid portion524 is substantially rectangular, with an enclosed top 528 and sides 529a-d and an open bottom 530 for receiving filter wheel 521. Opposedflanges 531 extend downward from one pair of opposed sides 529 b,d ofremovable lid portion 524 to support hub structure 522. Filter wheel 521is rotatably mounted through its center on hub structure 522.

Static base portion 523 holds removable lid portion 524 and filter wheel521. Static base portion 523 is substantially rectangular, with anenclosed bottom 532 and sides 533 a-d and an open top 534 for receivingfilter wheel 521. Opposed slots 535 extend downward into one pair ofopposed sides 533 b,d of static base portion 523 to receive opposedflanges 531. Opposed posts 536 extend upward from the other pair ofopposed sides 533 a,c of static base portion 523 to be received byopposed holes 537 in opposed sides 529 a,c of removable lid portion 524.Flanges 531 and slots 535, and posts 536 and holes 537, individually andcollectively form a post-to-hole mating structure that aligns staticbase portion 523 and removable lid portion 524 when the two portions aremated together to form the wheel case. Captive screws 538 situated inholes 537 and accessible from top 528 may be threaded into posts 536 tohold together removable lid portion 524 and static base portion 523.Static base portion 523 further may be fixed to an instrument platformto form a portion of a light source module, detector module, or otheroptical assembly, among other applications.

The assembled wheel case is substantially light-tight, except for lightthat is transmitted through two sets of opposed windows 539 included instatic base portion 523. Windows 539 are used for transmitting lightthrough the wheel case and through a selected optical filter containedin a filter cartridge 525 in filter wheel 521. Windows 539 are locatedon opposite sides of hub structure 522, so that any given optical filterin filter wheel 521 can be rotated into alignment with either set ofwindows. In turn, light sources, detectors, and other optical componentscan be aligned with either or both sets of filters. Generally, the wheelcase includes at least one set of windows, which may be located on thestatic portion, removable portion, or other portion of the wheel case.

Filter wheel 521 may be rotated by a drive motor 540, which is attachedto removable lid portion 524 in optical filter wheel assembly 520. Drivemotor 540 or other driver mechanisms also may be operatively connectedto optical filter wheel assembly 520 at other points and in othermanners.

FIG. 28 also shows a mechanism by which optical filter wheel assembly520 may be disassembled and reassembled. Optical filter wheel assembly520 is disassembled as follows. First, any associated instrument ispowered down and unplugged. Second, any secondary housing enclosingoptical filter wheel assembly 520 is removed. Third, drive motor 540 isunplugged at its inline connector 541. Fourth, captive screws 538 areloosened. Finally, removable lid portion 524 and filter wheel 521 arepulled out of static base portion 523.

Optical filter wheel assembly 520 may be reassembled as follows. First,filter cartridges 525 are checked to verify that they are properlyseated in filter wheel 521, and filter wheel 521 is checked to verifythat it rotates smoothly about hub structure 522 when moved by hand.Second, removable lid portion 524 and filter wheel 521 are inserted intostatic base portion 523, aligning flanges 531 with slots 535, and posts536 with holes 537. Third, captive screws 538 are tightened. Fourth,drive motor 540 is plugged back in at inline connector 541. Fifth, anysecondary housing is replaced. Finally, any associated instrument isplugged back in and powered up, if desired.

FIG. 29 shows a partially exploded perspective view of a removableportion 542 of an optical filter wheel assembly, including a filterwheel 543, removable lid portion 544, and drive motor 545. Filter wheel543 includes a set of “short” filter cartridges 546 and a set of “tall”filter cartridges 547. Filter wheel 543 may hold a variety of filtercartridges, so long as the filter cartridges are configured to fit inapertures 548 in the filter wheel. Generally, opposed apertures infilter wheel 543 should contain matching filter cartridges or a suitableslug to balance the filter wheel and to prevent unfiltered radiationfrom reaching a detector.

FIG. 29 also shows a mechanism by which short filter cartridges 546 maybe removed and replaced. Generally, short filter cartridges 546 includean optical filter 549 permanently affixed by suitable means, such asglue, to a short filter barrel 550 having a low profile. Optical filter549 may include an intensity filter, a spectral filter, or apolarization filter, among others. Short filter cartridges 546 areremoved from filter wheel 543 as follows. First, with the filter wheelremoved as described above, the desired short filter cartridge islocated by sight or by location. (Filter cartridge locations within thefilter wheel may be marked on the filter wheel or elsewhere forreference.) Second, the short filter cartridge is removed by turning itcounter-clockwise, which unscrews it. The short filter cartridge may beturned by hand or by a special tool, such as a spanner wrench 551 havingprongs 552 that engage grooves 553 in the sides of the short filtercartridge 554. Finally, filter changes are noted on the filter wheel orelsewhere and in any associated instrument software. Short filtercartridges 546 may be replaced in filter wheel 543 by reversing theprocess, turning the short filter cartridge clockwise.

FIG. 30 shows a partially exploded perspective view of a removableportion 555 of an optical filter wheel assembly, as shown in FIG. 29.FIG. 30 also shows a mechanism by which tall filter cartridges 556 maybe removed and replaced. Generally, tall filter cartridges 556 includean optical filter 557 affixed by a removable friction member 558 to atall filter barrel 559. Optical filter 557 may include an intensityfilter, a spectral filter, or a polarization filter, among others.Friction member 558 and tall filter barrel 559 may be substantiallyannular. Tall filter cartridges 556 may be removed from and replaced infilter wheel 560 much like short filter cartridges 546; however, tallfilter cartridges 556 generally are turned by hand rather than by atool.

FIGS. 31 and 32 show a perspective view of a mechanism by which opticalfilters may be replaced in the tall filter cartridges. First, as shownin FIG. 31, the optical filter 561 is placed in the tall filter barrel562. Optical filter 561 should be oriented properly if one side isdifferent than the other. Additional optical filters 561 can be placedin tall filter barrel 562, if desired. Second, as shown in FIG. 32, afunnel structure 563 is placed on top of tall filter barrel 562. Third,an annular friction member 564 is placed in funnel structure 563,followed by a slug 565. Slug 565 and optical filter 561 haveapproximately equivalent peripheral dimensions, including radii. Fourth,slug 565 is pushed down through funnel structure 563 to compressfriction member 564, which should fit snugly against optical filter 561.Finally, slug 565 and funnel structure 563 are removed. The completedtall filter cartridge then can be installed in a filter wheel, asdescribed above.

Optical filter 561 also may be replaced by other techniques. Generally,the tall filter cartridges incorporate a mechanism that permits easyreplacement of different optical filters in the same cartridge,enhancing the flexibility of the tall cartridges.

Optical filter 561 may be removed from the tall filter cartridge asfollows. First, a lint-free cloth is placed on a work surface. Second,the installed optical filter 561 (or slug 565) is pushed gently near itscenter with a gloved finger or thumb, which will cause the opticalfilter 561 and friction member 564 to drop out of tall filter barrel562. Removed optical filter 561 should be stored so that it will notbecome dirty or scratched.

FIGS. 33 and 34 show detailed views of a short filter cartridge 566,which includes a short filter barrel 567 and optical filter 568. Shortfilter barrel 567 is substantially annular, with a threaded lowerportion 569 that screws into an aperture in a filter wheel, and agraspable upper portion 570 having a knurled rim 571 that may be turnedby hand. Optical filter 568 is supported by upper portion 570, andmounts adjacent a stop structure 572 and inner wall 573 on short filterbarrel 567, so that it is substantially centered relative to shortfilter barrel 567. Stop structure 572 includes an edge 574 orientedsubstantially perpendicular to a principal plane of optical filter 568and to inner wall 573.

FIGS. 35 and 36 show detailed views of a tall filter cartridge 575,which includes a tall filter barrel 576 and optical filter 577. Tallfilter cartridge 575 resembles short filter cartridge 566 in manyrespects. Tall filter barrel 576 is substantially annular, with athreaded lower portion 578 that screws into an aperture in a filterwheel, and a graspable upper portion 579 having a knurled rim 580 thatmay be turned by hand. Optical filter 577 is supported by upper portion579, and mounts adjacent a stop structure 581 and inner wall 582. Stopstructure 581 includes an edge 583 oriented substantially perpendicularto a principal plane of optical filter 577 and to inner wall 582. Innerwall 582 may be substantially perpendicular to the optical filter, ashere, or it may have a funnel portion that graduates in diameter in adirection toward the stop structure, among other configurations. Lowerportion 569 of short filter barrel 567 is substantially identical tolower portion 578 of tall filter barrel 576. However, upper portion 570of short filter barrel 567 is shorter than upper portion 579 of tallfilter barrel 576, giving it a lower profile. In addition, opticalfilter 568 of short filter barrel 567 is permanently affixed to upperportion 570, whereas optical filter 577 of tall filter barrel 576 isremovably sandwiched in upper portion 579 between stop structure 581 anda friction member 584. Friction member 584 holds optical filter 577 inplace relative to inner wall 582 in tall filter cartridge 575 by staticfriction, without any thread, groove, or adhesive. For this reason,among others, optical filters of various numbers and sizes may besecured.

Friction member 584 may take a variety of forms, including acompressible ring having an uncompressed outer diameter greater than theinner diameter of inner wall 582. The compressible ring may exert aforce on the inner wall that provides sufficient static friction to holdan optical filter snugly in place during routine use, while alsopermitting easy removal when replacing optical filters.

FIGS. 37 and 38 show detailed views of a funnel structure 585, which isused for loading an optical filter into a tall filter cartridge or otherholder as described above. Funnel structure 585 is substantially annularand includes inner and outer walls 586, 587 and a top end 588 and loweredge 589. Lower edge 589 includes a groove 590 adjacent inner wall 586configured to rest on top of a filter cartridge or other holder. Theinner diameter of funnel structure 585 measured between inner walls 586enlarges gradually in a direction from lower edge 589 to top end 588.

FIG. 39 shows a partial perspective view of an alternative filter holderassembly 592. Filter holder assembly 592 includes an elongate filtercartridge 593 and a base 594. Elongate filter cartridge 593 includes afilter end 593 a and a pivot end 593 b. Filter end 593 b is configuredto hold optical filters, and includes two filter slots 593 c in whichoptical filters 595 may be glued or otherwise attached. Generally, thefilter end may hold one or more optical filters, using slots, apertures,short or tall filter cartridges, or other mechanisms. Filter slots maybe left open so that light passes unfiltered, or filter slots may befilled with filters so that light is filtered, or filled with slugs orother opaque structures so that light is blocked. Pivot end 593 b isconfigured turnably to attach to a hub structure, and includes anaperture 593 d for receiving a drive axle or other pivot structure.Generally, the pivot end may attach through any means to any suitabledrive mechanism. Elongate filter cartridge 593 is fan shaped, filter end593 a being wider than pivot end 593 b, although other shapes also arepossible.

Base 594 generally supports elongate filter cartridge 593. Base 594includes a hub structure 596 and major and minor walls 594 a,b thatsubstantially surround elongate filter cartridge 593 on all but oneside. Elongate filter cartridge 593 is turnably attached at its pivotend 593 b to hub structure 596 through a drive axle 597, about which itmay turn. Base 594 also includes a window 594 c in major wall 594 a.

Elongate filter cartridge 593 may be used for moving an optical filterin and out of an optical path, much like a filter wheel or filter slide,by turning elongate filter cartridge 593 about hub structure 596.Because elongate filter cartridge 593 may move one or a few filters inand out of an optical path by turning through a limited angle, it may beconfigured to require less space than a filter wheel of comparableradius. A drive mechanism 598 may be controlled or base 594 may beconfigured to limit the angle through which elongate filter cartridge593 may turn. For example, in filter holder assembly 592, a position 594d on minor wall 594 b forms a stop structure that physically limitsmovement if drive mechanism 594 d attempts to turn elongate filtercartridge 593 past the wall.

Sample Transporter

FIGS. 40-43 show a stage, which generally comprises any mechanism forsupporting a composition in a sample container for analysis by theanalyzer. In analyzer 50, the stage includes a transporter 600 and baseplatform 700.

FIGS. 40-42 show transporter 600, which includes a transporter body 602and substantially parallel first and second transporter flanges 604 a,bthat extend outward from transporter body 602. First and secondtransporter flanges 604 a,b terminate in first and second transporterextensions 606 a,b that turn in toward one another without contactingone another. Transporter extensions 606 a,b may be joined by a connectorportion 607. Transporter body 602, flanges 604 a,b and extensions 606a,b lie substantially in a plane and define a transporter cavity 608that is larger than the expected peripheral dimension of any samplecontainers which the transporter is intended to support. The shape ofthis cavity is chosen to accommodate the shape of the preferred samplecontainers. In analyzer 50, cavity 608 is generally rectangular toaccommodate generally rectangular sample containers, such asmicroplates. In analyzer 50, long sides of the rectangular samplecontainer are positioned against flanges 604 a,b.

Transporter 600 includes a shelf structure and associated framestructure for supporting a microplate or other sample container. Forexample, transporter shelves 610 along portions of body 602, flanges 604a,b and extensions 606 a,b form a shelf structure that supports thebottom of the sample container. The shelf structure also could includeother support mechanisms, such as pins or pegs.

The transporter also includes an automatic sample container positioningmechanism 620 for positioning sample containers precisely andreproducibly within cavity 608. Mechanism 620 includes Y and X axispositioning arms 622 a,b that contact the sample container to controlits Y and X position, respectively. Here, a Y axis is defined asgenerally parallel to transporter flanges 604 a,b and an X axis isdefined as perpendicular to the Y axis and generally parallel totransporter extensions 606 a,b. Other coordinate systems also can bedefined, so long as they include two noncolinear directions.

Y-axis positioning arm 622 a lies substantially within a channel 624 inbody 602. Y-axis positioning arm 622 a includes a rod 626 a which isbent at substantially right angles to form three substantially coplanarand equal-lengthed segments. A first end segment 628 a of rod 626 aterminates near cavity 608 in a bumper 632 for engaging a samplecontainer. A second end segment 634 a of rod 626 a terminates away fromcavity 608 in an actuator tab 636 a for controlling movement of arm 622a. Actuator tab 636 a is bent away from body 602. First and second endsegments 628 a, 634 a are substantially parallel. A middle segment 638 aof rod 626 a connects the two end segments at their nontabbed ends 640,641. An X-axis biasing spring 642 a having first and second spring ends644, 648 is slipped over rod 626 a. First spring end 644 is held tosecond end segment 634 a of rod 626 a by a clamping-type retaining ring650. Second spring end 648 rests against a rod bearing 652. The Y-axisbiasing spring extends substantially parallel to first and second endsegments 628 a, 634 a. The force from spring 642 a is transmitted to rod626 a by the clamping action of retaining ring 650.

X-axis positioning arm 622 b also lies substantially within channel 624in body 602 and is similar to Y-axis positioning arm, except that (1)first end segment 628 b is longer and middle segment 638 b is shorter inrod 626 b of the X-axis positioning arm than in rod 626 a of the Y-axispositioning arm, (2) first end segment 628 a terminates in a lever tab653 in the X-axis positioning arm rather than in bumper 632 in theY-axis positioning arm, and (3) the two rods bend in opposite directionsbetween first end segments 628 a,b and second end segments 634 a,b.

X-axis positioning arm 622 b is connected via lever tab 653 to an X-axispositioning lever 654 that lies along transporter flange 604 b. X-axispositioning lever 654 includes first and second lever projections 656,658 and is pivotally mounted about a lever pivot axis 659 to transporter600 near the intersection of body 602 and flange 604 b. First leverprojection 656 is substantially perpendicular to flange 604 b and abutslever tab 630 b on X-axis positioning arm 622 b for actuating thepositioning lever. Second lever projection 658 also is substantiallyperpendicular to flange 604 b and includes an edge 660 for contacting asample container.

Transporter 600 functions as follows. For loading, the transporteroccupies a loading position substantially outside a housing. In thisposition, actuator tabs 636 a,b abut an actuator bar 670, shown in FIG.43. In addition, biasing springs 642 a,b are compressed, and bumper 632and second projection 658 having edge 660 are pulled out of cavity 608.A person, robot, or mechanical stacker then can place a sample containerinto cavity 608 so that the bottom of the sample container rests onshelves 610. Cavity 608 is larger than the sample container tofacilitate this placement and to accommodate variations in samplecontainer size.

In some configurations, connector portion 607 may be removed, such thattransporter 600 has an open end. This open end permits a microplatetransfer device to enter cavity 608 and the generally rectangular areaof the holder. The microplate transfer device may, after moving into thegenerally rectangular area, move down relative to transporter 600,thereby gently placing the microplate into the generally rectangulararea.

For reading, the transporter must deliver the sample container to anexamination site inside the housing. In this process, the transportermoves parallel to second end segments 634 a,b and actuator tabs 636 a,bdisengage actuator bar 670. Biasing spring 642 a pushes Y-axispositioning arm 622 a toward cavity 608. Bumper 632 engages the samplecontainer and pushes it away from body 602 until it abuts extensions 606a,b. Biasing spring 642 b pushes X-axis positioning arm 622 b towardcavity 608. Edge 660 of second projection 658 engages the samplecontainer and pushes it away from flange 604 b until it abuts flange 604a.

As long as the sample container is placed in any position on the lowerguide shelves, it may be positioned (registered) precisely andreproducibly against a reference corner 672 within cavity 608 under theaction of both positioning arms. Biasing springs 642 a,b can be chosento have different strengths, so that the X-Y positioning action isperformed less or more forcefully. In analyzer 50, middle segment 638 band first lever projection 656 of positioning lever 654 can be varied inlength to cause registration to occur in series, first along the X-axisor first along the Y-axis, and second along the Y-axis or second alongthe X-axis, respectively. For example, reducing the length of middlesegment 638 b and reducing the length of projection 656 will causeregistration to occur first in the X-axis, and second in the Y-axis.

Positioning lever 654 and bumper 632 are retracted when body 602 of theautomatic microplate positioning transporter is moved to the ejectposition by the X, Y stage. Thus, the microplate is placed ontransporter shelf 610 only when the lever and bumper are retracted. Twosprings 642 a,b are attached to the rods, which run along the length ofthe transporter body and end perpendicular to the body. When thetransporter is moved to the eject position, the two perpendicular endsof the rods encounter a stop 670, which consists of a rectangularstructure located above and parallel to the body. The stop prevents thetwo perpendicular ends of the actuators, and thus the actuators, frommoving with the transporter body. This causes the two springs tocontract, changing the position of the transporter arms and increasingthe amount of room for the microplate. The microplate then can be placedon the guide shelf of the body. When the body of the automaticmicroplate positioning transporter is moved back away from the stop, thetwo perpendicular ends of the actuators no longer are blocked, whichallows the actuators, springs, and transporter arms to move into theiroriginal position. The expansion of the springs pushes the microplateexactly into position, as defined by the reference corner.

Thus, components of transporter 600 act as first and second releasableclamp mechanisms. The first releasable clamp mechanism applies a forceagainst a first (e.g., Y or X) side of the microplate, thereby securingthe microplate in the holder. The second releasable clamp mechanismapplies a force against a second (e.g., X or Y) side of the microplate,thereby securing the microplate in the holder from two sides. Theseclamp mechanisms may sandwich a microplate between the positioning armsand opposing portions of the frame structure, such that the positioningarms function as pushers and the opposing portions of the framestructure function as bumpers for the clamp mechanisms.

The invention provides a method of automatically feeding microplates inand out of an analyzer. The method comprises (1) automaticallydelivering a microplate just outside an opening to the analyzer, (2)moving a gripping device from inside the analyzer, through the opening,to a location immediately below the microplate; and (3) gently placingthe microplate onto the gripping device. The method further may compriseclamping the microplate in the holder by applying a first force againsta first side of the microplate, applying a second force against a secondside of the microplate, and/or serially performing the clamping steps.

FIG. 43 shows a base platform 700 with drive mechanisms for moving atransporter 702 between loading and examination positions or sites. Aspreviously described, transporter 702 includes flanges 704 a,b defininga cavity 706 for receiving and gripping a microplate (not shown). AY-axis drive mechanism 707 is provided for moving transporter 702 alonga first track 708 relative to the Y-axis, from a loading position 710toward an examination position 712. An X-axis drive mechanism 713 isprovided to move transporter 702 to examination position 712 along asecond track 714 relative to the X-axis.

In operation, a microplate is loaded in transporter 702 at loadingposition 710. Transporter 702 is driven toward the examination positionby Y-axis drive mechanism 707. A sensor (not shown) detects the presenceof the sample container. The analyzer may be configured automatically toread the microplate once the sensor detects its presence, or theanalyzer may be configured to signal the system controller through adata port that a microplate has been received and that the analyzer isready to accept a command to begin reading. The X- and Y-axis drivemechanisms then operate together to align selected microplate wells withan optical axis, substantially parallel to a Z-axis, along which asensed volume for luminescence detection may be defined by opticalcomponents contained in one or both of a top and bottom optics headpositioned above and below base platform 700, respectively.

Transporter 700 thus may function both as a sample delivery device inand out of the analyzer, and as a moveable stage for supporting thesample container at the examination site. The cavity in the transporterpermits analysis to be carried out from below the holder, when thetransporter is functioning as a stage at the examination site.

X- and Y-axis drive mechanisms 707 and 713 may be controlled by ahigh-performance motion control system that maximizes throughput whileminimizing detection errors. A preferred high-performance control systemincludes precision five-phase stepper motors that employ encoderfeedback to move the microplate quickly and accurately to each readposition. The control system may optimize the acceleration/decelerationprofiles of the microplate to minimize shaking of fluid within themicroplate, for example, by minimizing “jerk” (the time rate of changeof the acceleration of the microplate). Alternatively, the controlsystem may increase throughput by moving plates more quickly, if highervariation in results due to increased shaking and setting time may betolerated.

Exterior Features

FIG. 44 shows a high-throughput luminescence analyzer 50 constructed inaccordance with the invention. Components of the analyzer are maintainedin a housing 800, both for organization and for protection. Housing 800is substantially rectangular and includes light-tight exterior top 802,side 803 a-d, and bottom walls 804 that reduce background inluminescence measurements. The walls may include vents 806 to facilitateair flow through the analyzer and a transporter port 807 for sampleinput/output. Housing 800 also may include feet 808 to support theanalyzer and to permit air flow between the analyzer and any supportstructure on which the analyzer is placed.

Analyzer 50 is substantially automated. The analyzer is designed so thatuser interactions occur primarily through a control unit 810, anelectronic input/output panel 812, and a break-out box (not shown), eachof which supports a variety of input/output functions. The analyzer alsois designed so that sample input/output occurs primarily through atransporter/stage 814 and an optional sample feeder 816.

Transporter 814 generally comprises any device for supporting a samplecontainer, as described above. In analyzer 50, transporter 814 movesbetween the interior and exterior of the analyzer, and may be used aloneor together with sample feeder 816 for sample input/output.

Sample feeder 816 generally comprises any device for automaticallyprocessing multiple samples, as described below. In analyzer 50, samplefeeder 816 includes a first (input) station 818 for holding samplecontainers to be read, a third (output) station 820 for holding samplecontainers that have been read, and a second (direct transporter access)station 822 for inputting or outputting sample containers that bypassesthe input and output stations. Input and output stations 818, 820accommodate preprocessing and postprocessing sample containers bins 824,826 that hold and organize stacks of sample containers before and afterreading, respectively. Sample feeder 816 also may include a barcodereader 828 for automatically identifying labeled sample containers.

The sample container generally comprises any container for holding atleast one sample. Preferred sample containers include microplates. Othersuitable sample containers include any sample containers having a shapeand rigidity suitable for processing in an analyzer, such as slides orsupported gels.

Control Unit

Control unit 810 generally comprises any interface used for directinput/output functions. The control unit may be integrated into theanalyzer, or it may be a separate unit that can be positioned away fromthe analyzer or affixed to the analyzer at one or more locations. Thecontrol unit also may include more than one unit, each dedicated todifferent input/output functions or to use at different locations.

The control unit 810 may be used in conjunction with a host computer fora variety of input/output functions. For example, the control unit maybe used to input commands, such as signals to start and stop theinstrument. Similarly, the control unit may be used to display outputinformation, such as instrument status, instrument diagnostics,measurement results, and other information generated by the analyzer indifferent assay modes. The control unit is especially useful forautomated operations that require manual user intervention.

FIG. 45 shows an enlarged isolated view of control unit 810 of analyzer50. Control unit 810 is a separate unit that statically or swivelablyaffixes to the analyzer at any one of a plurality of docking locations.Control unit 810 is substantially L-shaped, with substantiallyperpendicular inner surfaces 830 a,b that mate with adjacentsubstantially perpendicular walls of the analyzer including top wall 802and one of side walls 803 a-d although other shapes are possible. In itspreferred orientation, control unit 810 is mounted so that front face832 is substantially parallel with one of side walls 803 a-d of analyzer50.

Control unit 810 includes various data input and output components.Front face 832 includes a gas-plasma display 834, keypad 836, andindicator lights 838. Control unit 810 also may include additionaland/or alternative components, and their relative organization maydeviate from that shown in the drawings and discussed below. Gas-plasmadisplay 834 is located in the upper center of front face 832 and is usedto provide messages regarding instrument status. Additional displaysand/or alternative display formats, such as light-emitting diodes (LEDs)and liquid crystal displays (LCDs), also may be used.

Keypad 836 is located below and to the right of gas-plasma display 834and includes four keys. A “start” key 840 initiates the sample-readingprocess. A “load/eject” key 842 loads or ejects a sample container, suchas a microplate, depending upon the current status of the instrument. A“reset” key 844 reinitializes the instrument, sending motors to theirhome positions and turning off the audible alarm. A “status” key 846alters the state of a continuous light source or activates reversestack. Additional keypads and additional and/or alternative keys alsomay be employed. Alternative methods of data entry, such as a computermouse or touch screen, also may be employed.

Indicator lights 838 are located to the left of the display and keypad.A “power” light 848 indicates that power is being supplied to theinstrument. A “service” light 850 indicates that a service procedure isneeded, such as changing a light source. A “fault” light 852 indicatesthat a critical fault has occurred, which is a fault that requiresintervention by an operator. Additional and/or alternative indicatorlights also may be provided.

Control unit 810 also may include audio signals. For example, an audiblealarm within the interior of control unit 810 may sound in the event ofa critical fault. Alternative audio signals, such as prerecorded orsynthesized voice messages, also may be used.

Control unit 810 may be moved between at least two control interfacedocking-panel mounting locations 854 a,b on the instrument. A firstdocking location 854 a is located near an upper edge of sample inputside 803 b of housing 800. This configuration is especially suitable formanual operation, because control unit 810 and transporter port 807 arepositioned on the same side of analyzer 50. A second docking location854 b is located near an upper edge of back side 803 d of housing 800.This configuration is especially suitable for robotic operation, becausecontrol unit 810 and transporter port 807 are positioned on oppositeside of analyzer 50, facilitating robotic access to transporter port807. Such flexible positioning permits commands to be entered and statusinformation, diagnostic information, measurements, and other informationto be read from multiple positions. Flexible positioning is especiallyconvenient when one or more sides of the analyzer are blocked due toanalyzer placement or nearby peripherals. Alternatively, it permits twoor more control units to be connected at once, increasing convenienceand flexibility.

FIG. 46 shows a control interface docking location 860. Control unit 810includes an electronic connector prong, which can be mated with anelectronic connector port 862 at docking location 860. Electronicconnector port 862 is connected to a host computer, allowing thecomputer to communicate with the control unit, so that a user cancontrol the analyzer by inputting information through the control unit.Electronic connector port 862 preferably includes an RS-232 serial port,and preferably is connected to the host computer through an RS-232cable. Control unit 810 also includes other mating structure, includingsubstantially cylindrical prongs that match with receptors 864 andlatches 866, and indentations that match with dimples 868, at dockinglocation 860. Positioning docking location 860 at sites 854 a,b on topwall 802 of housing 800 reduces the stress on the mating structure whenthe control unit is mounted; however, docking location 860 also can bepositioned at other sites on or off housing 800.

Input/output Panel

The input/output panel generally comprises any ports used for basicinput/output functions. These include ports for providing andcontrolling power input to the analyzer, and for inputting andoutputting data and commands. Components of the input/output panel maybe collected for convenience in one location or positioned at variouslocations on the analyzer.

FIG. 47 shows an enlarged isolated view of control input/output panel812. In analyzer 50, input/output panel 812 includes a power switch 870,power entry module 872, auxiliary port 874, and two RS-232 serial ports876. Power switch 870 is located in the left center of the panel and isused to actuate analyzer 50. Power entry module 872 is located below thepower switch and is used to supply power to analyzer 50; power arrivesvia a standard electrical cord 878 that may be plugged into a wallsocket. Auxiliary port 874 and serial ports 876 are located above and tothe right of the power entry module and are used for input/output. Theseports provide flexibility, because they permit the analyzer tocommunicate with several different peripherals. Additional power entrymodules and additional and/or alternative communication ports forinput/output in alternative formats and positions also may be used. Amodel/regulatory label 880 containing written information regarding theanalyzer is provided below power entry module 872 on the input/outputpanel.

Break-Out Box

The analyzer also may include an external “break-out” accessory boxconnected to the instrument with a cable. The break-out box may includea connection block that allows the analyzer to provide a general purposeand hard-wired electrical interface to external devices, such as lamps,warning alarms, enunciators, associated instruments, and external systemcontrollers. Through the break-out box, the instrument's software can beprogrammed to send or receive control signals from external systems orto control or provide signals to external devices. These control signalscan be conditioned on the occurrence of predetermined internal events,such as when the analyzer finishes reading a plate or when a fault suchas a mechanical jam occurs. Through the break-out box, the instrumentalso can accept signals from external devices or controllers to startreading a plate or perform other programmable functions.

Sample Feeder

FIGS. 48-50 show a sample feeder 948, which generally comprises anymechanism for automatic processing of multiple sample containers. Samplefeeder 948 enhances convenience by reducing the amount of humanintervention required to run the analyzer. Sample feeder 948 alsoenhances throughput by reducing the amount of time required to processmultiple sample containers.

Generally, sample feeder 948 operates as follows. Before reading, arobot (1) removes a sample container from the bottom of an input stackof sample containers at an input station, (2) transports the samplecontainer to a direct transporter access station, and (3) transfers thesample container to a transporter. After reading, the robot (1) takesthe sample container from the transporter, (2) transports the samplecontainer to an output station, and (3) transfers the sample containerto the bottom of an output stack of sample containers. Sample feeder 948requires only two motors to provide these functions with high throughput(˜5 seconds for load and unload time).

FIG. 48 shows sample feeder 948 with its preprocessing andpostprocessing bins removed, so that internal mechanisms can be viewed.A microplate 949 is loaded from the bottom of a stack of microplates inthe input bin into a first (input) station 950. Microplate 949 then istransported on a tray (not shown) to a second (direct transporteraccess) station 952, where the microplate is handed off to a transporter(not shown). The transporter transports microplate 949 generally alongan axis 953 to an examination site inside the analyzer. After analysis,the transporter transports microplate 949 back along axis 953 generallyin the opposite direction to second station 952. Microplate 949 then ishanded back to the tray, and transported to a third (output) station954, where the microplate is added to the bottom of a stack ofmicroplates in an output bin.

In analyzer 50, a first linear path defined by axis 953 connects theexamination site to the second station, and a second linear pathconnects the first second and third stations, wherein the first linearpath is substantially perpendicular to the second linear path. However,analyzer 50 also may have other configurations. For example, theexamination site and the first, second, and third stations may all bepositioned along a single substantially linear path.

In input station 950, a combination of two lifters and four latchescooperate to singulate or pick a single microplate from the bottom of astack. (These lifters are concealed by microplate 949 in FIG. 48.)Latches 958 have pick portions that extend into the cavity of firststation 950 and support a stack of microplates. Latches 958 are disposedtoward the microplates by configuring the latch to have a center ofgravity above and inward relative to a pivot point. As the lifters areraised in the input station, the pick portions of the latches are pushedout of the way, so that the microplate can be supported and lowered bythe lifters. After one microplate has passed below the latch, latches958 move back into a supporting position relative to the remainder ofthe stack.

In output station 954, a different latch configuration is employed.Latches 960 are urged inward toward the microplates by a spring (notshown). When lifter 962 lifts a microplate against latches 960, themicroplate pushes the latches out of the way. After one microplate haspassed above the latch, latches 960 move back into a supporting positionrelative to the remainder of the stack.

FIGS. 49A and 49B show how input station 950 operates. FIG. 49A showsmicroplate 949 as it is being picked up at input station 950 prior toanalysis. Lifters 970 have moved up through holes in tray 972 to contactthe bottom of microplate 949, and in the process have pushed latches 958out of the way. FIG. 49B shows the same structures as FIG. 49A, exceptthat lifters 970 have dropped, thereby lowering microplate 949 onto tray972 for transport to the analyzer. Pick portions of latches 958 havemoved back into the cavity to support the remainder of the stack.

FIGS. 50A and 50B show how output station 954 operates. FIG. 50A showsmicroplate 949 after it has been delivered to output station 954following analysis. Lifters 962 then move through holes in tray 972 toraise microplate 949 toward a stack of microplates in the output bin(not shown). FIG. 50B shows the same structures as FIG. 50A, except thatlifters 962 have raised microplate 949 past latches 960. Latches 960 arespring biased toward the cavity of third station 954. As lifters 962raise microplate 949, latches 960 are pushed out of the way by the outercontour of microplate 949. Once microplate 949 is above latches 960, thelatches return to their inward position to support the stack ofmicroplates in the output bin. Lifters 962 then retreat downwardcompletely out of the holes in tray 972, so that the tray can translateback to input station 950 to collect another microplate for delivery tothe analyzer.

FIG. 51 shows how lifter 962 operates. Generally, the lifter comprisesany mechanism configured to raise or lower a sample container. Lifter962 is substantially rectangular and includes top 974, side 975, andbottom 976 walls. Each of an opposed pair of side walls 975 includes twosloped drive channels 978, which function as cams, and a verticalguidance channel 980. In sample feeder 948, pins are inserted into drivechannels 978 and guide channel 980. In alternative embodiments, pins andchannels may be replaced with other components, including ridges,bearings, or rollers. Pins inserted into drive channels 978 areconnected to a drive motor, which moves the pins through drive channels978 between a top position A nearer top wall 974 and a bottom position Bnearer bottom wall 976. The pins move horizontally along a line 982, sothat the pins push against a side 984 of drive channels 978, urginglifter 962 to move both horizontally and vertically. Pins inserted intoguidance channels 980 are connected to relatively fixed portions ofsample feeder 948, preventing horizontal motion, but permitting verticalmotion, so that lifter 962 only moves vertically. As the pin movesbetween positions A and B, the pin moves a horizontal distance H and avertical distance V. It is the vertical displacement that creates theraising and lowering motions. H and V may be optimized for particularsample containers and travel distances; in sample feeder 948, H and Vare optimized for microplates and are approximately 10 cm and 3.5 cm,respectively. Lifter 962 is raised when the pin is near position A, andlifter 962 is lowered when the pin is near position B.

In use, the drive motor moves the pins horizontally at a substantiallyuniform rate; consequently, the slope of drive channel 978 determinesthe mechanical advantage and the rate of vertical motion. Near positionsA, B, and an intermediate position C, the slope of drive channel 978 issubstantially zero, so that there is substantially no vertical motion.Stated differently, near positions A, B, and C, a preselected verticalposition corresponds to a range of horizontal positions. Thisconfiguration makes the vertical position relatively insensitive tomotor precision or manufacturing tolerance, because the lifter will beat the same vertical position whenever it simply is near positions A, B,or C. Between positions A and C, and between positions B and C, theslope of drive channel 978 is nonzero, so that there is vertical motion.The slope is largest (approximately 30°) between positions A and C, sothat the lifter raises and lowers relatively rapidly when it is farthestfrom the bottom of the stack of sample containers. The slope is smallest(approximately 15°) between positions B and C, so that the lifter raisesand lowers relatively slowly when it is nearest to the bottom of thestack of sample containers.

The drive motor generally comprises any mechanism configured to generatea driving motion. The drive motor used in sample feeder 948 is a steppermotor, which generates a constant torque. Generally, stepper motors andcams provide alternative mechanisms for performing the same function, inthis case, generating a varying rate of motion. However, pairing astepper motor and cam together in the invention provides severaladvantages. In particular, the cam provides mechanical advantage andpositional insensitivity, and permits the stepper motor to be run at aconstant, optimal speed. If the stepper motor were used alone, anelectronic control system would be necessary to vary raising andlowering speed. Conversely, if the cam were used alone, with anonstepper motor, an electronic control system with feedback controlwould be necessary to vary raising and lowering speed.

Together, the lifters and latches form a singulation mechanismconfigured to separate a microplate (or other sample container) from astack of microplates in the down-stacking or input operation. Thismechanism has inherently low sensitivity to the exact size, shape,construction material, and surface finish of the microplate. Asdescribed, the invention may include four inwardly sloping, tapered (orangled) latches that cause the stack of microplates to self-centerwithin the microplates input area to accommodate both relatively smalland large microplates sizes. Also as described, the invention mayinclude a feature that causes the microplates to drop gently when thesingulation mechanism disengages from the edges of the microplates, thusallowing the microplates to drop onto the lifter mechanism supportstructure, which lowers the microplates to the tray without spillingfluid from the wells.

The down-stacking latches pivot on pins and are actuated by the liftermechanism so as to retract when the lifter mechanism rises, therebyreleasing the bottom microplate from the stack and allowing it to dropsoftly onto the lifter. When the latches retract, they pivot on theirsupport pins such that their centers of gravity are offset.Consequently, when the lifter mechanism is lowered, the latches will beactivated by gravity to return to their nonretracted or extended state,thereby preventing the next microplates in the stack from dropping asthe lifter mechanism is lowered. Because the offset in the center ofgravity of the latches is only enough to cause them to return to theirextended position, they press only very lightly on the edges of themicroplate as it drops. Because the ends of the latches are polishedsmooth, they exert only a small frictional force on the edges of themicroplates so as not to cause the microplate to tilt or otherwise hangup as the lifter mechanism is lowered and the microplate is placed onthe tray.

Together, the lifters and latches also form a stacking mechanismconfigured to add a microplate to a stack of microplates. Generally, theup-stacking mechanism resembles the down-stacking mechanism. The liftermechanism raises the microplate by a fixed amount, thereby causing it topass by four spring-loaded latches, which retract as the microplate israised by the lifter. Once the bottom of the microplate is above the topof the latch, the latches are released, and a spring on each latchcauses the latch to extend under the microplate. The lifter mechanismthen is lowered, causing the microplate to be captured by the nowextended latches. The up-stacked microplate thus is added to the bottomof the output stack.

Sample feeder 948 also may employ alternative singulation mechanisms.For example, singulation mechanisms may (1) take microplates from thebottom of the stack in the input station and add microplates to thebottom of the stack in the output station, as above, (2) takemicroplates from the bottom of the stack in the input station and addmicroplates to the top of the stack in the output station, (3) takemicroplates from the top of the stack in the input station and addmicroplates to the bottom of the stack in the output station, or (4)take microplates from the top of the stack in the input station and addmicroplates to the top of the stack in the output station.

Sample feeder 948 permits a robot to deliver a sample container to theinput station and to retrieve a different sample container from theoutput station, both in the same trip. This feature is known as “processcompression” and reduces robot hand travel in servicing analyzer 50. Forexample, if there were only one loading station (e.g., the transporter),the robot would have to remove the analyzed microplate before deliveringthe unanalyzed microplate. Thus, process compression replaces twoseparate robot movements with one robot movement. Sample feeder 948 maybe configured so that the input and output stations can hold amicroplate to facilitate process compression.

Sample feeder 948 is designed to be flexible. The input and outputstations can accommodate a variety of commercially available microplatesand are large enough to allow microplates to be placed in them by arobot or a human hand. Suitable microplates typically have 96 or 384wells, but other configurations also can be accommodated. The input andoutput stations also can accommodate a variety of commercially availablepreprocessing and postprocessing microplate bins for holding a stack ofmicroplates before and after analysis, respectively. Preprocessing binsmay be removed from the input station and replaced with anotherpreprocessing bin containing a new stack of microplates with samples tobe analyzed. Similarly, postprocessing bins positioned may be removedfrom the output station and replaced with another postprocessing bin toreceive a new stack of microplates with samples that have been analyzed.Microplate bins may be used with other robotics to dispense, wash, andread without restacking microplates. Suitable microplate bins typicallycan accommodate 0-60 microplates.

Sample feeder 948 also may include a barcode reader, as shown in FIG.48, which can be used automatically to identify labeled microplates. Thebarcode reader 986 preferably is positioned in either of two positionsadjacent direct transporter access station 952; these positions permitbarcode reader 986 to read barcodes mounted on the long edge or theshort edge of microplates. Barcodes are read when sample feeder 948moves the microplate from input station 950 to direct transporter accessstation 952. Barcodes cannot be read when microplates are delivereddirectly to the direct transporter access station 952. Barcode reader986 can be programmed to decode a variety of symbologies, including SPC(EAN, JAN, UPC), Code 39 (3-43 digits), Codabar (3-43 digits), Standard2 of 5 (3-43 digits), Interleaved 2 of 5 (4-43 digits), Code 93 (5-44digits), and MSI-Plessey (4-22 digits), among others. Informationobtained from the barcode can be used for various purposes. For example,the barcode can be used to name the report file. The barcode also can beused to convey instructions to the analyzer relating to required changesin assay mode or optics configuration.

Analyzer Set-Up Calibration, and Reading

Operation of the analyzer includes set-up, calibration, and reading.Setup of the analyzer includes selection of an assay mode and selectionof optical components and conditions to optimize performance in thatassay mode. Selection of optical components and conditions requiresknowledge of the assay mode, microplate, fluid level, total fluidvolume, and sensed volume, among other parameters. Optical componentsmay be changeable manually or automatically, depending on the component.For example, the size of the sensed volume may be adjusted manually byreplacing the fiber optic cables adjacent the examination area, andmanually or automatically by changing the apertures in front of thefiber optic cables. Similarly, the position of the sensed volume mayadjusted manually, or automatically by scanning a positive control wellor wells to obtain the maximum signal given the average fluid level inthe wells. Manually changeable components may include standard or“quick-change” components.

Calibration of the analyzer may include using a calibration plate. Acalibration plate may be shaped like a microplate and include featuresthat can be manually, optically, mechanically, and/or electronicallyrecognized. For example, a calibration plate may include preciselylocated apertures, mirrors, light sources (such as light-emitting diodes(LEDs)), and/or fluorescent reference standards to verify that theoptics, detection, and positioning systems are operating properly.

Reading by the analyzer may be performed in five phases. Phase 1comprises loading a microplate in the transporter. During this phase, aperson, robot, or microplate feeder mechanism places the microplate onthe microplate transporter of the X,Y stage. A computer-controlled X,Ymicroplate registration mechanism ensures that microplates have thecorrect alignment relative to the optics beam.

Phase 2 comprises sensing the microplate in the transporter. During thisphase, a sensor is activated that tells the local or system controllerthat the microplate has been delivered. The local controller can beginreading the microplate either after sensing the microplate or afterreceiving a command from the system controller to start reading.

Phase 3 comprises finding the top of the microplate. During this phase,the top of the microplate is found with the top-of-the-plate sensorlocated in the optics head, followed by computer-controlled adjustmentof the Z-position of the optics head.

Phase 4 comprises reading the microplate. During this phase, themicroplate is moved automatically from well to well to allow analysis ofthe contents of each well by use of a high performance motion controlsystem with preselected acceleration/deceleration profiles and settingtimes to provide maximum possible throughput with minimum acceptableread error.

Phase 5 comprises unloading the microplate from the transporter.

Assay Modes

The analyzer may support a variety of assay modes, including (1)luminescence intensity, (2) luminescence polarization, (3) time-resolvedluminescence, (4) chemiluminescence, and (5) absorbance. Aspect of theseassay modes are described below to show the versatility and sensitivityof the analyzer. Additional assays and/or alternative methods forperforming the described assays also may be employed in conjunction withthe analyzer provided by the invention. Additional information regardingthese assay modes may be found in U.S. Provisional Patent ApplicationSerial No. 60/082,253, filed Apr. 17, 1998, and incorporated herein byreference.

Luminescence Intensity Mode

Luminescence intensity measurements use a continuous light source. Lightproduced by the light source is routed through a luminophore-specificexcitation filter and a low-luminescence fiber optic cable to the opticshead. A beamsplitter splits the light, reflecting light into the assaywell and transmitting light into a light monitor. The light monitorchecks the light source continuously and can be programmed to alert theuser if the light source fails. Light emitted from the assay well maypass back through the beamsplitter and then is routed through a fiberoptic cable to an emission filter that conditions the light beforedetection by a photomultiplier tube.

The analyzer may use confocal optics elements to direct excitation lightinto the assay well and to detect light emitted from the well, all froma sensed volume that may be small compared to the overall volume of thewell. Because the sensed volume does not change with the volume of theassay well, performance in different microplates is virtually identical.Z-position within the well may be set manually or automatically. Forhomogeneous assays, the location with the highest signal-to-noise (S/N)ratio and highest signal-to-background (S/B) ratio typically is in themiddle of the well. For cell-based assays, the location with the highestS/N and S/B ratio typically is at the bottom of the well, whereluminescence from the cells is maximized and luminescence from the fluidis minimized. Conditions that optimize the S/N and S/B ratios may bedetermined empirically.

Luminescence intensity measurements may be made from either the top orbottom of the sample well. Bottom reading delivers a higher signal thantop reading because the bottom focal area is larger, but bottom readingalso delivers a lower S/N ratio because microplates or other samplecontainers typically autoluminesce.

The user has fill control of analyzer settings through software. Forluminescence measurements, the user selects the excitation and emissionfilters, top or bottom reading, and read time. Optional parametersinclude the magnitude and duration of plate shaking, well-to-well settletime, and Z-height adjustments.

Luminescence Polarization Mode

Luminescence polarization measurements use the same opticalconfiguration as luminescence intensity measurements, except thatpolarization measurements always employ emission and excitationpolarization filters and the top optics head. Light from a continuouslight source, preferably a xenon-arc source, is routed through anexcitation filter, low-luminescence fiber optic cable, and apolarization filter, which typically is in the S orientation. Abeamsplitter then splits the light, reflecting polarized light into theassay well and transmitting light into the light monitor. Light emittedfrom the assay well may pass back through the beamsplitter and then isrouted through a fiber optic cable to an emission and polarizationfilter (in either the S or P orientation) that conditions the lightbefore detection by a photomultiplier tube.

The analyzer makes two measurements for each assay well, one withexcitation and emission polarizers aligned and one with excitation andemission polarizers crossed (as described above). Either polarizer maybe static or dynamic, and either polarizer may be set to be S or P.

The continuous light source preferably comprises a high-intensity,high-color temperature light source, such as a xenon arc lamp. Such alamp minimizes photon noise and hence reduces reading time at a givennoise level. When combined with the optimized luminescence detectionsystem, the continuous high-intensity light source increases lightthroughput and decreases background.

As in luminescence intensity mode, confocal optics elements may directthe excitation light into a small sensed volume in a selected region ofthe well. The best S/N ratio typically is obtained from the middle ofeach well, because spurious polarization signals from luminophores boundto the well surfaces is minimized. Conditions that optimize the S/N andS/B ratios may be determined empirically.

For luminescence polarization measurements, the user selects theexcitation and emission filters, and read time. Optional parametersinclude the magnitude and duration of plate shaking, well-to-well movetime, and Z-height adjustments.

Time-Resolved Luminescence Mode

Time-resolved luminescence measurements use substantially the sameoptical configuration as luminescence intensity and luminescencepolarization measurements, except that time-resolved luminescencemethods use the upper optics head and the substitution of a flash lamp,preferably a xenon flash lamp, for a continuous lamp as the lightsource. The flash lamp creates a brief flash of excitation light, whichis followed by time-dependent luminescence. Time-dependent measurementsmay be delayed to avoid short-lifetime autoluminescence, and hastened toavoid long-lifetime autoluminescence, if desired.

As in luminescence intensity mode, confocal optics elements may directthe excitation light into a small sensed volume in a selected region ofthe well. The location of the sensed volume can be changed using theZ-height parameter. The optimal S/N and S/B can best be determinedempirically.

For time-resolved luminescence, the user selects the excitation andemission filters, delay time, integration time, and cycle time. Optionalparameters include the magnitude and duration of plate shaking,well-to-well settle time, and Z-height adjustments.

Chemiluminescence Mode

Chemiluminescence measurements use a dedicated read head andphotomultiplier tube adjacent the top optics head and separate fromthose used in photoluminescence measurements. Light emitted from anassay well is collected through a specially-baffled read head andaperture that reduce well-to-well cross-talk. Collected light then isrouted through a low-luminescence fiber optic cable to an optimizedphotomultiplier tube having relatively low dark counts and a blue-greenshifted response.

Alternatively, chemiluminescence measurements may use thephotoluminescence optical system, especially if it is desirable to sensechemiluminescence from a sensed volume within the sample container. Toreduce background in this mode, the light source module in thephotoluminescence system may be “parked” between detectors, so that theassociated floating head assembly abuts only a solid surface.

For luminescence measurements, the user can select read time. Optionalfeatures include plate shaking, well-to-well settle time, and Z-heightadjustments.

Absorbance Mode

Absorbance measurements require a combination of top illumination andbottom detection, or bottom illumination and top detection, and may usecontinuous or flash light sources.

Measurement Modes

The analyzer may support a variety of measurement modes for detectingluminescence, including (1) photon counting, (2) current integration,and (3) imaging modes. Aspect of these measurement modes are describedbelow to show the versatility and sensitivity of the analyzer.Additional measurement modes and/or alternative methods for performingthe described measurement modes also may be employed in conjunction withthe analyzer provided by the invention.

Photon-Counting Mode

Transmitted light may be detected in photon-counting mode. In thisapproach, the photons comprising the detected light are counted, andintensity is reported as the number of counted photons per unit time.Photon counting is well-suited for assays with low light levels, becauseeach photon is individually reported. Conversely, photon counting isill-suited for assays with high light levels, because the detector maybecome saturated and unable to distinguish the arrival of one photonfrom the arrival of more than one photon. Suitable detectors forpracticing this method include PMTs.

Current-Integration Mode

Transmitted light also may be detected in current-integration mode. Todecrease the average read time per well, the electronics can beconfigured to integrate the detector current resulting from theluminescence signal until a preset threshold is achieved. This isequivalent to collecting light from the well until a predeterminednumber of photons are collected. The component of the signal-to-noiseratio due to the photon noise of the emission light then will be equalto the square root of the number of photons collected by the detector.This feature is implemented using an integrating current-to-voltageconverter at the detector output coupled to an analog comparator inparallel with an analog-to-digital converter. At the beginning of eachmeasurement cycle, the integrator is reset and the time required for theintegrated detector current to trip the comparator is measured. Theintegration time is a representation of the number of photons collectedand hence the signal level. If the signal is too small to cause thecomparator to be tripped within the maximum time allowed for theintegration, the analog-to-digital converter is used to digitize thevoltage appearing at the output of the integrator. Because the value ofthe integration capacitor and the voltage across it both are known, thenumber of photons collected can be calculated by taking the product ofthe integration capacitance and the measured voltage and dividing it bythe electronic charge (1.602×10⁻¹⁹ Coulombs per electron). Suitabledetectors for practicing this method include PMTs.

Imaging Mode

In addition to analysis of single wells, this invention also supportssimultaneous reading of many wells located in a fixed area of amicroplate. Large-area fiber optic bundles and an imagingcharged-coupled device (CCD) detector make it possible to excite anddetect a fixed area of the microplate at once. Using this method, thedetection limit and time to read a microplate is constant regardless ofthe number of wells on the microplate as long as the fiber size in thebundle is small compared to the smallest well to be measured (e.g., >4fibers per well) and the CCD pixel size is small compared to the fibersize (e.g., >4 pixels per fiber). If the fiber optic bundle is randomlyoriented, a calculation procedure can be used during setup to map eachCCD pixel to a specific location on the microplate. For example, asingle microplate well containing a fluorescent compound can be used tomap the CCD pixels through the fiber bundle to the microplate surface byrepositioning the well repeatedly to include all CCD pixels.

The above description elaborates on the general architecture of theinvention, while also describing preferred embodiments. Other relatedembodiments are possible and may be desirable for specific applications.For example, it may be desirable to commercialize only a portion of thepreferred embodiment to meet the needs of different customers orspecific markets. Also, the preferred embodiments provide for anexpandable architecture wherein the light sources and detectors can beadded as required to provide new assay modalities, or to take advantageof new types of light source and detectors, as they become commerciallyavailable. For example, blue LEDs have become commercially availableonly in the last few years, and blue laser diodes are expected to becomecommercially available within the next few years. The architecture ofthe invention is designed to be flexible so as to allow incorporation ofnewly commercialized technology with the goal of making such technologyavailable to high-throughput screening laboratories at the earliestpossible date.

Another alternative embodiment may include a plurality of confocaldetection systems mounted in a linear array or matrix. A linear array of8 or 12 confocal detectors may be used with one or more light sourcesand 8 or 12 detectors to simultaneously detect an entire row or columnof a 96 well microplate. The same detectors could also be used to read384 or 1536 well plates with the proper aperture installed since thewell-to-well pitch of the hedger density plates are evenly divisibleinto that of the 96 well plate. In another example, the confocaldetection systems could be mounted in an n-by-m array and could alsodetect one or more plate formats.

What is claimed is:
 1. An apparatus for detecting light transmitted froma sample, the apparatus comprising a stage for supporting a sample in amicroplate well having a diameter X a light source positioned to deliverlight to the sample, a detector positioned to receive light transmittedfrom the sample, and a confocal optics device that positions a focalspot in the sample, the focal spot having a diameter of at least about0.3X.
 2. An apparatus for detecting light transmitted from a sample, theapparatus comprising a stage for supporting a sample in a microplatewell having a diameter X, a light source positioned to deliver light tothe sample, a detector positioned to receive light transmitted from thesample, and an optical relay structure that positions a sensed volume inthe sample, the sensed volume being characterized by a waist regionhaving a diameter D and a Z-pick-up having a height of at least about2D.
 3. An apparatus for detecting light transmitted from a sample, theapparatus comprising a stage for supporting a sample in a microplatewell, the sample having boundary interfaces defined by walls of themicroplate well and an upper gas interface, a light source positioned todeliver light to the sample, a detector positioned to receive lighttransmitted from the sample, a sample handling mechanism capable ofautomatically detecting light serially from microplate wells in adensity of at least about 384 wells per plate, and an optical relaystructure that positions a sensed volume in the sample, the sensedvolume being spaced away from the boundary interfaces.
 4. The apparatusof claim 3, wherein the sample handling mechanism is capable ofautomatically detecting light serially from microplate wells in adensity of at least about 1536 wells per plate.
 5. An apparatus fordetecting light transmitted from a composition, the apparatus comprisinga stage configured to support the composition in an examination site, anautomated registration device that automatically brings successivecompositions and the examination site into register for successiveanalysis of the compositions, a light source positioned to deliver lightto the composition in the examination site along an excitation opticalaxis, and a detector positioned to receive light transmitted from thecomposition in the examination site along an emission optical axis,wherein the excitation optical axis and the emission optical axis arenot colinear.
 6. The apparatus of claim 5, wherein the excitationoptical axis is angled relative to the emission optical axis.
 7. Theapparatus of claim 6, wherein the excitation optical axis is angled tocreate total internal reflection in the composition.
 8. A lightdetection system comprising a stage for supporting a microplate having aplurality of wells, each well having a maximum volume capacity of about55 microliters or less, a light source, a detector, an optical relaystructure that transmits light from the light source to a samplecontained in a well in the microplate, and from the sample to thedetector, and a Z-axis adjustment device that alters location of asensed volume inside the sample to optimize signal-to-backgroundperformance of the system.
 9. The system of claim 8, wherein themicroplate has at least about 384 wells.
 10. The system of claim 8,wherein each well in the microplate has a maximum volume capacity ofabout 20 microliters or less.
 11. The system of claim 8, wherein eachwell in the microplate has a substantially frustoconical shape.
 12. Thesystem of claim 8, wherein the optical relay structure includes confocaloptics.
 13. The system of claim 8, wherein the optical relay structuregenerates a sensed volume in the sample that substantially avoidsboundary interfaces of the sample.
 14. The system of claim 8, whereinthe optical relay structure generates a sensed volume that extractsmaximum signal from the sample.
 15. The system of claim 8, wherein theoptical relay structure generates a sensed volume, at least a portion ofthe sensed volume having a shape that is substantially frustoconical.16. The system of claim 8, wherein the optical relay structure generatesa sensed volume, at least a portion of the sensed volume having a shapethat substantially corresponds to the shape of the well.
 17. The systemof claim 8, wherein the microplate has a frame portion that iscompatible with robotics that are designed to handle standardmicroplates having 96 wells per plate.
 18. An apparatus for detectinglight transmitted from a composition, the apparatus comprising means forsupporting the composition in an examination site, the composition beingcontained in a spatial volume delineated by boundary interfaces, meansfor automatically bringing successive compositions and the examinationsite into register for successive analysis of the compositions, meansfor producing light and delivering the light to the composition in theexamination site, means for transmitting light substantially exclusivelyfrom a sensed volume of the composition, means for automaticallypositioning the sensed volume substantially away from at least one ofthe boundary interfaces of the composition, and means for detecting thelight transmitted substantially exclusively from the sensed volume ofthe composition.
 19. The apparatus of claim 18, wherein the successivecompositions are held in adjacent wells in a microplate.
 20. Theapparatus of claim 18, wherein the means for producing light includes alight source selected from the group consisting of flash lamps, arclamps, incandescent lamps, fluorescent lamps, electroluminescencedevices, lasers, laser diodes, and light-emitting diodes (LEDs).
 21. Theapparatus of claim 18, wherein the means for transmitting lightsubstantially exclusively from a sensed volume include a confocal opticselement.
 22. The apparatus of claim 18, wherein the means forpositioning the sensed volume automatically positions the sensed volumebetween the boundary interfaces according to stored parameters relatingto the type of plate and the type of assay being performed.
 23. Theapparatus of claim 18, wherein the means for positioning the sensedvolume automatically positions the sensed volume after sensing theboundary interface.
 24. The apparatus of claim 18, wherein the means forpositioning the sensed volume automatically positions the sensed volumeto maximize the signal detected from the composition.
 25. The apparatusof claim 18, wherein the means for positioning the sensed volume adjuststhe relative positions of the sensed volume and at least one boundaryinterface by moving the composition relative to portions of the meansfor transmitting light from the sensed volume.
 26. The apparatus ofclaim 18, wherein the means for positioning the sensed volume adjuststhe relative positions of the sensed volume and at least one boundaryinterface by moving portions of the means for transmitting light fromthe sensed volume relative to the composition.
 27. The apparatus ofclaim 18, wherein the means for detecting light includes a detectorselected from the group consisting of photomultiplier tubes (PMTs),photodiodes, avalanche photodiodes, charge-coupled devices (CCDs), andintensified CCDs.
 28. An apparatus for detecting light transmitted froma sample, the apparatus comprising a stage for supporting a sample in amicroplate well, the sample having boundary interfaces defined by wallsof the microplate well and an upper gas interface, a light sourcepositioned to deliver light to the sample, a detector positioned toreceive light transmitted from the sample, and an optical relaystructure that focuses a non-diffraction-limited spot into the samplewithout illuminating significantly any of the boundary interfaces of thesample.
 29. The apparatus of claim 28, wherein the optical relaystructure includes a confocal optics arrangement.
 30. The apparatus ofclaim 28 further comprising a Z-axis optical adjustment device thatautomatically adjusts positions of the sensed volume relative to theboundary interfaces.
 31. The apparatus of claim 30, wherein the Z-axisoptical adjustment device automatically positions the sensed volumebetween the boundary interfaces according to stored parameters relatingto the type of plate and the type of assay being performed.
 32. Theapparatus of claim 30, wherein the Z-axis optical adjustment deviceautomatically positions the sensed volume after sensing the boundaryinterface.
 33. The apparatus of claim 30, wherein the Z-axis opticaladjustment device adjusts the relative positions of the sensed volumeand at least one boundary interface by moving the composition relativeto portions of the optical relay structure.
 34. The apparatus of claim30, wherein the Z-axis optical adjustment device adjusts the relativepositions of the sensed volume and at least one boundary interface bymoving portions of the optical relay structure relative to thecomposition.
 35. The apparatus of claim 28, wherein the microplate wellhas a diameter D, the spot having a diameter of at least about 0.3D. 36.The apparatus of claim 28 further comprising a sample handling mechanismcapable of automatically detecting light serially from microplate wellsin a density of at least about 384 wells per plate.
 37. An apparatus fordetecting light transmitted from a sample, the apparatus comprising astage for supporting a sample in a microplate well having a shape thatis substantially frustoconical, a light source positioned to deliverlight to the sample, a detector positioned to receive light transmittedfrom the sample, and an optical relay structure located between thelight source and the detector, the optical relay structure includingconfocal optics that create a sensed volume in the sample, at least aportion of the sensed volume having a shape conforming substantially tothe shape of the well.
 38. The apparatus of claim 37, wherein the sensedvolume is characterized by a waist region having a diameter D and aZ-pick-up having a height of at least about 2D.
 39. The apparatus ofclaim 37, wherein the microplate well has a diameter X, the sensedvolume being character sized by a waist region having a diameter of atleast about 0.3X.